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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. Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes.

    Science.gov (United States)

    Novak, Jakub; Cerny, Ondrej; Osickova, Adriana; Linhartova, Irena; Masin, Jiri; Bumba, Ladislav; Sebo, Peter; Osicka, Radim

    2017-09-24

    Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

  4. 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

  5. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

    OpenAIRE

    Donato, Gina M.; Goldsmith, Cynthia S.; Paddock, Christopher D.; Eby, Joshua C.; Gray, Mary C.; Hewlett, Erik L.

    2012-01-01

    B.pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B.pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B.pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b an...

  6. Plant adenylate cyclases.

    Science.gov (United States)

    Lomovatskaya, Lidiya A; Romanenko, Anatoliy S; Filinova, Nadejda V

    2008-01-01

    Adenylate cyclase (AC) (ATP diphosphate-lyase cyclizing; EC 4.6.1.1) is a key component of the adenylate cyclase signaling system and catalyzes the generation of cyclic adenosine monophosphate (cAMP) from ATP. This review summarizes data from the literature and the authors' laboratory on the investigation of plant transmembrane (tmAC) and soluble (sAC) adenylate cyclases, in comparison with some key characteristics of adenylate cyclases of animal cells. Plant sAC has been demonstrated to exhibit similarities with animal sAC with respect to certain characteristics. External factors, such as far-red and red light, temperature, exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin exert a significant influence on the activity of plant tmAC and sAC.

  7. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.

    Science.gov (United States)

    Donato, Gina M; Goldsmith, Cynthia S; Paddock, Christopher D; Eby, Joshua C; Gray, Mary C; Hewlett, Erik L

    2012-02-17

    Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  8. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

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    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  9. 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.

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

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    Masin, Jiri; Osicka, Radim; Bumba, Ladislav; Sebo, Peter

    2015-11-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

  11. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-10

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

  13. 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.

  14. Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

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    Hewlett, E L; Gray, M C; Ehrmann, I E; Maloney, N J; Otero, A S; Gray, L; Allietta, M; Szabo, G; Weiss, A A; Barry, E M

    1993-04-15

    Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin

  15. 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; 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.

  16. 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.

  17. Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

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    Cannella, Sara E.; Ntsogo Enguéné, Véronique Yvette; Davi, Marilyne; Malosse, Christian; Sotomayor Pérez, Ana Cristina; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Ladant, Daniel; Chenal, Alexandre

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins. PMID:28186111

  18. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism.

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    Kaminski, N E; Koh, W S; Yang, K H; Lee, M; Kessler, F K

    1994-11-16

    Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation

  19. 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

  20. Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway.

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    Portugal, Leivi; Muñóz-Garay, Carlos; Martínez de Castro, Diana L; Soberón, Mario; Bravo, Alejandra

    2017-01-01

    Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K(+) ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K(+) ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

  1. 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...

  2. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  5. Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase.

    Science.gov (United States)

    Inhorn, L; Fleming, J W; Klingberg, D; Gabig, T G; Boswell, H S

    1988-04-01

    Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3-independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the "differentiation" agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G-proteins appear to play a secondary role.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. Thyrotropin receptor-adenylate cyclase function in human thyroid neoplasms.

    Science.gov (United States)

    Saltiel, A R; Powel-Jones, C H; Thomas, C G; Nayfeh, S N

    1981-06-01

    The action of thyrotropin (TSH) on plasma membranes was studied to elucidate the mechanism of hormonal regulation of malignant versus normal human thyroid tissue. Thyroid plasma membranes of six specimens of papillary or follicular carcinoma and six of adenoma, as well as adjacent normal tissue obtained from these patients, were evaluated with respect to binding of 125I-labeled TSH and stimulation of adenylate cyclase. Scatchard analysis of TSH binding revealed the presence of two species of binding sites in normal thyroid of different affinities and capacities. In 11 of 12 tumors studied, the high-affinity binding site remained intact; however, the total number of low-affinity sites was markedly lower than normal tissue. Other parameters of binding were not altered in neoplastic thyroid. In each of these tissues, the hormone responsiveness and kinetics of adenylate cyclase activation were essentially identical to those observed in normal tissue, although basal activity was typically greater in the neoplasm. One carcinoma was totally deficient in both 125I-labeled TSH binding and TSH-stimulatable adenylate cyclase, although basal activity was detected. Furthermore, adenylate cyclase of this specimen was not activated by prostaglandin, in contrast to normal thyroid and other thyroid tumors. These results suggest that: (a) clinical behavior of thyroid carcinomas may not be reflected by TSH receptor-adenylate cyclase function; (b) lack of clinical response as manifest by tumor regression cannot be ascribed to the absence of functional TSH receptors or adenylate cyclase; and (c) decreased low-affinity binding present in tumors is not correlated with altered hormone responsiveness of adenylate cyclase but may reflect more general cancer-induced changes in membrane structure or composition.

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

    Directory of Open Access Journals (Sweden)

    Yasmeen Faiz Kazi

    2012-06-01

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

  12. 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.

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    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.

  13. Effect of mitomycin C on the activation of adenylate cyclase in rat ascites hepatoma AH130 cells.

    Science.gov (United States)

    Miyamoto, K; Matsunaga, T; Sanae, F; Koshiura, R

    1986-09-01

    Isoproterenol (IPN)-stimulated activity of adenylate cyclase was enhanced in a dose-dependent manner by exposure of AH130 cells to mitomycin C (MMC). The enhancement was also observed in prostaglandin E1-, guanine nucleotide analog-, NaF-, cholera toxin- and forskolin-stimulated activities of the enzyme but not in manganese-stimulated activity. In addition, even when the cells pretreated with islet-activating protein were exposed to MMC, IPN-stimulated activity of adenylate cyclase was enhanced. Anaerobic exposure of AH130 cells to MMC somewhat inhibited IPN-stimulated activity of adenylate cyclase in contrast with aerobic exposure. Exposure of cells to adriamycin also caused enhancement of IPN-stimulated activity of adenylate cyclase but exposure to nitrogen mustard inhibited the enzyme stimulation by IPN. The enhancing effect of MMC was lost by the combined treatment with alpha-tocopherol. From these results, it was shown that MMC modulated the activity of adenylate cyclase, probably through alterations in membrane structure.

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

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    Ma’ayan Israeli

    2016-08-01

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

  15. Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

    Science.gov (United States)

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Takagi, K; Satake, T; Hasegawa, T

    1989-02-01

    Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

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

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    Tomoya eNakamachi

    2012-11-01

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

  17. Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.

    Science.gov (United States)

    Colombo, Sonia; Paiardi, Chiara; Pardons, Katrien; Winderickx, Joris; Martegani, Enzo

    2014-05-01

    Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.

  18. 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.

  19. Neurohypophyseal hormone-responsive renal adenylate cyclase. IV. A random-hit matrix model for coupline in a hormone-sensitive adenylate cyclase system.

    Science.gov (United States)

    Bergman, R N; Hechter, O

    1978-05-10

    A "random-hit" matrix model is proposed to account for the dynamic and steady state relationship between occupation of bovine renal medullary membrane receptors by [Lys8]vasopressin (LVP) and neurohypophyseal hormones (NHH) and the associated activation of membrane-bound adenylate cyclase. The model was developed by systematic introduction of specific rules concerning receptor coupling into a general structural model which consists of two square matrices of identical size, one composed of homogeneous R ("receptor") units, the second of homogeneous C ("cyclase") units. R units are either occupied (RO) or unoccupied (RU); C units are either active (CA) or inactive (CI). Hormone molecules are envisioned to "collide" with R units randomly; collision with RU leads to "binding", and occupation is maintained for a characteristic mean occupancy time, TO. In this structure, each R unit has an "interaction field" which consists of the "twin" unit in the "C" matrix, and the 4 nearest neighbor C units surrounding the twin. Occupation of an R unit leads to activation of all CI units in the interaction field of that R; CA units in the interaction field are refractory. Thus binding at a given R may "recruit" a variable number of inactive neighboring C units (5, 4, 3, 2, 1, or 0). The model requires that there be individual coupling delays between the moment of binding at a given R and subsequent activation of CI units (mean coupling delay (Td) approximately 10% To). Activation of C units persists as long as the "parent" R is occupied and is maintained for an additional short time interval (Tp) after RO reverts to RU, corresponding to hormone dissociation from receptor. The model accounts for the following previously demonstrated relations between LVP occupation of receptors and adenylate cyclase activation in bovine renal medullary membranes: 1) the shape of the nonlinear steady state relation between normalized (percentage maximal) receptor occupation (O) and cyclase activation

  20. [The aspects of adenylate cyclase activity regulation in myocardium cell membranes during hypokinesia].

    Science.gov (United States)

    Bulanova, K Ia; Komar, E S; Lobanok, L M

    1999-01-01

    Nonstimulated and isoproterenol, GTF, GITF, NaF stimulated activities of the adenylate cyclase in sarcolemma in white rats' myocardium was studied after two weeks of hypokinesia. As was established, in restrained animals the sensitivity of adenylate cyclase to the specified agents was increased and transition to the bimodal GTF regulation took place. It is hypothesised that involvement of membrane-bound Gi-proteins in the adrenergic effects on cardiomyocytes is one of mechanisms of the cardiotropic effects of restraint and heart distresses.

  1. Regulation of adenylate cyclase of Dictyostelium discoideum by divalent cations and adenosine analogs

    Energy Technology Data Exchange (ETDEWEB)

    Khachatrian, L.; Howlett, A.; Klein, C.

    1986-03-05

    Cyclic AMP is synthesized and secreted in a pulsatile fashion as a chemotactic signaling system intrinsic to the differentiation program of D. discoideum. They examined the regulation of D. dischoideum adenylate cyclase using a membrane fraction which exhibits high specific activity enzyme. When Mn-ATP was used as substrate, increasing Mn/sup 2 +/ concentrations activated the enzyme 3 to 8 fold. In contrast, Mg ion increased the adenylate cyclase activity by only 60%. These results suggested an activation of the catalytic subunit by Mn/sup 2 +/. Inhibition of activity was observed in response to adenosine and its analogs. P-site agonist, 2',5'-Dideoxy-adenosine, inhibited activity by about 25% in the presence of Mg/sup 2 +/, and about 80% in presence of Mn/sup 2 +/. This inhibition was not dependent on guanine nucleotides. The data are in agreement with characteristics of P-site regulation of the catalytic subunit of eukaryotic systems. Kinetic analysis of previously reported inhibition of D. discoideum adenylate cyclase by guanine nucleotides revealed that guanine nucleotides do not compete for the substrate binding site. Further, the enzyme activity cannot be accounted for by guanylate cyclase. Their data suggest that regulation of adenylate cyclase may exist not only at the catalytic subunit but also via inhibitory G protein, N/sub i/.

  2. 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.

  3. 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.

  4. 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...

  5. 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.

  6. Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

    Science.gov (United States)

    Sulakhe, P V; Leung, N L; Arbus, A T; Sulakhe, S J; Jan, S H; Narayanan, N

    1977-01-01

    1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity. PMID:18147

  7. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    Energy Technology Data Exchange (ETDEWEB)

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  8. 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.

  9. 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.

  10. Cyclic 3', 5'-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase

    OpenAIRE

    1983-01-01

    In Dictyostelium discoideum, binding of cAMP to high affinity surface receptors leads to a rapid activation of adenylate cyclase followed by subsequent adaptation within several minutes. The rate of secretion of [ 3H ]cAMP, which reflects the state of activation of the enzyme, was measured. Caffeine noncompetitively inhibited the response to cAMP. Inhibition was rapidly reversible and pretreatment of cells with caffeine for up to 22 min had little effect on the subsequent responsiveness to cA...

  11. Differential Effects of Temperature on cAMP-induced Excitation, Adaptation, and Deadaptation of Adenylate and Guanylate Cyclase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of ~1 s; excitation of adenylate c

  12. Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase.

    Science.gov (United States)

    Agarwal, Nisheeth; Lamichhane, Gyanu; Gupta, Radhika; Nolan, Scott; Bishai, William R

    2009-07-02

    With 8.9 million new cases and 1.7 million deaths per year, tuberculosis is a leading global killer that has not been effectively controlled. The causative agent, Mycobacterium tuberculosis, proliferates within host macrophages where it modifies both its intracellular and local tissue environment, resulting in caseous granulomas with incomplete bacterial sterilization. Although infection by various mycobacterial species produces a cyclic AMP burst within macrophages that influences cell signalling, the underlying mechanism for the cAMP burst remains unclear. Here we show that among the 17 adenylate cyclase genes present in M. tuberculosis, at least one (Rv0386) is required for virulence. Furthermore, we demonstrate that the Rv0386 adenylate cyclase facilitates delivery of bacterial-derived cAMP into the macrophage cytoplasm. Loss of Rv0386 and the intramacrophage cAMP it delivers results in reductions in TNF-alpha production via the protein kinase A and cAMP response-element-binding protein pathway, decreased immunopathology in animal tissues, and diminished bacterial survival. Direct intoxication of host cells by bacterial-derived cAMP may enable M. tuberculosis to modify both its intracellular and tissue environments to facilitate its long-term survival.

  13. Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus

    Science.gov (United States)

    Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus. PMID:28066725

  14. Adenylate-cyclase activity in platelets of patients with obsessive-compulsive disorder

    Directory of Open Access Journals (Sweden)

    D Marazziti, S Baroni

    2009-07-01

    Full Text Available D Marazziti, S Baroni, L Palego, I Masala, G Consoli, M Catena Dell’Osso, G Giannaccini, A LucacchiniDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Pisa, ItalyAbstract: Although the main biological hypothesis on the pathophysiology of obsessive-compulsive disorder (OCD is centered on the serotonin system, indications are available that other neurotransmitters, and even second messengers, particularly the cyclic adenosine monophosphate (cAMP signaling, may be involved, though effective data are few. Therefore, the aim of the present study was to evaluate and compare the basal and isoprenaline (ISO-stimulated velocity of adenylate-cyclase (AC in human platelet membranes of patients with OCD and healthy control subjects. The results showed that the basal and ISO-stimulated AC activity, as well as the dose-response curves of ISO by using agonist concentrations ranging between 0.1 nM and 10 µM, were not different in the two groups. However, OCD patients showed lower EC50 and higher Emax values than healthy subjects. These findings suggest the presence of supersensitive β-adrenergic receptors in platelets of OCD patients.Keywords: obsessive-compulsive disorder, norepinephrine, second messengers, adenylate-cyclase, platelets, isoprenaline, β-adrenergic receptors

  15. 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.

  16. The effect of adenylate cyclase stimulation on endocochlear potential in the guinea pig.

    Science.gov (United States)

    Doi, K; Mori, N; Matsunaga, T

    1990-01-01

    Forskolin, a diterpene extracted from Coleus forskohlii, is potentially an important tool for studying the modulation of ionic currents by cAMP because it stimulates adenylate cyclase in a variety of cells. We studied the effect of forskolin on cochlear potentials and found that its perfusion of the scala vestibuli (SV) to a concentration more than 10(-5) M and the scala tympani (ST) to more than 10(-4) M produced a reversible elevation of the endocochlear potential (EP) in a dose-dependent manner. The cochlear microphonics recorded simultaneously with the EP was not depressed during the EP elevation. A large negative EP was induced by anoxia following the SV perfusion with forskolin (2 X 10(-4) M). The results suggest that the EP elevation produced by forskolin does not result from the decrease in the negative component of EP but from the increase in the positive component of EP.

  17. 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.

  18. 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

    that PACAP was located in delicate varicose nerve fibers that were most abundant in the internal cervical os, where they mainly seemed to innervate blood vessels and smooth muscle cells. PACAP-38 and PACAP-27 (10(-10)-10(-6) M) caused a concentration-dependent relaxation of the spontaneous activity......The distribution, localization, and smooth muscle effects of pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in the human female genital tract. The concentrations of PACAP-38 and PACAP-27 were measured by radioimmunoassays, and both peptides were found throughout the genital...... of the nonvascular smooth muscle strips from fallopian tube and myometrium in vitro. Likewise, both peptides (10(-10)-10(-6) M) caused relaxation of nonrepinephrine (10(-6) M)-precontracted intramyometrial arteries. No effect of the PACAP sequences, PACAP-(6-27), PACAP-(16-38), and PACAP-(18-27), on fallopian tube...

  19. Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells.

    Science.gov (United States)

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Hasegawa, T; Takagi, K; Satake, T

    1989-09-01

    Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.

  20. 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.

  1. Responsiveness of adenylate cyclase to pituitary gonadotropins and evidence of a hormone-induced desensitization in the lizard ovary.

    Science.gov (United States)

    Borrelli, L; De Stasio, R; Bovenzi, V; Parisi, E; Filosa, S

    1997-07-01

    Gonadotropins (FSH and LH) affect several mammalian gonadal functions. In particular, FSH stimulates oogonial proliferation and oocyte growth, while LH regulates ovulation and progesterone secretion. In lacertilian reptiles, gonadal function is also regulated by pituitary gonadotropins, but which hormone controls ovarian activities and the mechanisms of action are unknown. The present study aimed to clarify mechanisms of action of pituitary gonadotropins on the ovary of Podarcis sicula (Lacertilia). The data demonstrate that mammalian gonadotropins FSH and LH produce a threefold stimulation of adenylate cyclase activity in follicular membranes, while hCG and TSH are less effective, causing a twofold increase in adenylate cyclase activity. Neurotransmitters such as dopamine, serotonin, and catecholamines have no effect on enzyme activity. The action of mammalian FSH and LH on the ovary mimics the effect of homologous hormones: in lizard ovaries incubated in vitro in the presence of isolated homologous pituitary glands, the intracellular cAMP level increased by 50% with respect to control ovaries. Mammalian gonadotropins appear homologous to lizard gonadotropin(s): Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian beta FSH and beta LH. Both homologous and heterologous desensitization of adenylate cyclase activity occurs in the lizard ovary. In fact, responsiveness of adenylate cyclase to gonadotropin stimulation is abolished in animals 2 hr after in vivo treatment with FSH. Sensitivity to gonadotropin stimulation is restored 2 weeks after the beginning of the in vivo treatment. Desensitization was also observed in ovaries incubated in vitro with mammalian FSH or with isolated pituitary glands.

  2. 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.

  3. Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

    Science.gov (United States)

    Masugi, Yohei; Tanese, Keiji; Emoto, Katsura; Yamazaki, Ken; Effendi, Kathryn; Funakoshi, Takeru; Mori, Mariko; Sakamoto, Michiie

    2015-12-01

    Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

  4. Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

    Science.gov (United States)

    Yamazaki, Ken; Takamura, Masaaki; Masugi, Yohei; Mori, Taisuke; Du, Wenlin; Hibi, Taizo; Hiraoka, Nobuyoshi; Ohta, Tsutomu; Ohki, Misao; Hirohashi, Setsuo; Sakamoto, Michiie

    2009-04-01

    Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

  5. 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.

  6. 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.

  7. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development

    Science.gov (United States)

    Antal, Maria Cristina; Bénardais, Karelle; Samama, Brigitte; Auger, Cyril; Schini-Kerth, Valérie; Ghandour, Said; Boehm, Nelly

    2017-01-01

    Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors. PMID:28122017

  8. How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

    Science.gov (United States)

    Woods, A S; Jackson, S N

    2013-05-15

    Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Published by Elsevier Ltd.

  9. Cytochemical localization of adenylate cyclase activity in heart tissue with cerium.

    Science.gov (United States)

    Schulze, W; Will-Shahab, L; Küttner, I

    1986-01-01

    Adenylate cyclase (AC) activity showed a doses depending inactivation of the basal activity and of the sodium fluoride stimulation by cerium in homogenates of unfixed and fixed guinea pig hearts. The isoproterenol and guanine nucleotide stimulation was not more than two times of the basal activity in glutaraldehyde-prefixed heart homogenates in the presence of 2 mmol/l CeCl3. The inactivation of the AC (activity) by cerium was less than in the presence of lead. Test tube experiments showed no differences in the precipitation of imidodiphosphate in comparison with inorganic phosphate. The substrate AMP-PNP was not spontaneously hydrolysed by 2 mmol/l CeCl3. Ultrastructural analysis of cytochemical incubation of glutaraldehyde-fixed slices and small pieces of guinea pig heart tissue showed fine-amorphous precipitations of reaction products localized along the plasma membrane of the sarcolemma, the nexuses of the intercalated discs and the T-tubule membranes. No precipitates were found neither on the junctional nor on other SR membranes. Nonspecific coarse and clumped precipitates have been detected in the intercellular space on components of the basal membranes. It was not able to demonstrate cytochemically stimulation of AC by hormones or by sodium fluoride. The localization of the basal AC activity in heart tissue seems to be better with cerium as capture agent than with lead. However, differences in the localization of the AC activity in heart tissue were not observed.

  10. Cellular localization of pituitary adenylate cyclase-activating peptide (PACAP) following traumatic brain injury in humans.

    Science.gov (United States)

    van Landeghem, Frank K H; Weiss, Thorsten; Oehmichen, Manfred; von Deimling, Andreas

    2007-06-01

    The pituitary adenylate cyclase-activating peptide (PACAP) is involved in many processes of the developing and mature central nervous system, such as proliferation, differentiation, apoptosis, neurotransmission, inflammation and neuroprotection. Alternative posttranslational processing of PACAP results in two biologically active, amidated 27- and 38-amino acid peptides termed PACAP27 and PACAP38. In the present study, we examined whether traumatic brain injury (TBI) affects cellular immunopositivity for PACAP27 and PACAP38. Patients (n = 55) were classified into three groups dependent on their survival time (under 24 h, between 24 h and 7 days and between 7 days and 99 days postinjury). PACAP27 and PACAP38 were expressed by neurons and glial cells in normal human neocortex (n = 10). Following TBI, the total number of PACAP27- and PACAP38-positive cells was significantly decreased for a prolonged survival period within the traumatized neocortex. In the pericontusional cortex, the number of cells expressing PACAP27 and PACAP38 was significantly increased at all survival times examined. Triple immunofluorescence examinations revealed a significant increase in the absolute numbers of GFAP-positive reactive astrocytes as well as a decrease in the CNP-positive oligodendrocytes, each coexpressing PACAP27 or PACAP38 in the contusional and pericontusional cortex. We hypothesize that the increase of glial PACAP immunoreactivity may be interpreted as part of a complex endogenous neuroprotective response in the pericontusional regions, but the precise role of PACAP following TBI is yet to be determined.

  11. 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.

  12. Molecular cloning and expression of a chicken pituitary adenylate cyclase-activating polypeptide receptor.

    Science.gov (United States)

    Peeters, K; Gerets, H H; Princen, K; Vandesande, F

    1999-08-25

    Although, since the isolation of pituitary adenylate cyclase-activating polypeptide (PACAP), a wealth of literature has been published describing its localization, binding sites, and biological activities in a variety of mammalian tissues, only very little is known about PACAP in avian species. Therefore, in order to find out the sites of actions of PACAP and to elucidate its physiological significance in birds, we identified a chicken PACAP receptor homologue of the mammalian type I receptors (PAC(1)-Rs). The chicken PACAP type I cDNA sequence was obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with 3'- and 5'-RACE PCR. This cDNA encodes a 471 amino acid precursor protein, sharing 81-83% sequence identity with mammalian analogs and 76% amino acid identity with the goldfish type I PACAP receptor. Northern blot analysis of chicken brain poly(A)(+)-rich RNA revealed the presence of a 5.5 kb and 7.5 kb PAC(1) receptor transcript. RT-PCR revealed that the chicken PACAP receptor is mainly expressed in the brain and gonads. A smaller amount of the receptor mRNA was found in pituitary, adrenal gland, kidney, intestine, pancreas, lung, and heart tissue. In situ hybridization with specific antisense oligodeoxynucleotide probes showed a widespread distribution of PAC(1) receptor mRNA in the chicken brain, with the highest expression being found in the dorsal telencephalon, olfactory bulb, hypothalamus, optic tectum, and cerebellar cortex. These findings suggest that PACAP affect a variety of functions both in the brain and peripheral tissues of the chicken.

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

    Directory of Open Access Journals (Sweden)

    Yan Dong

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

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

    Science.gov (United States)

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) 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 × 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 (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

  15. 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

  16. Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

    Science.gov (United States)

    Hamelink, Carol; Tjurmina, Olga; Damadzic, Ruslan; Young, W Scott; Weihe, Eberhard; Lee, Hyeon-Woo; Eiden, Lee E

    2002-01-08

    The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an "emergency response" cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.

  17. 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.

  18. 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.

  19. Morphine-induced changes of adenylate and guanylate cyclase in locus ceruleus, periaqueductal gray, and substantia nigra in rats.

    Science.gov (United States)

    Shijun, Hong; Liping, Zhao; Yongqiang, Qu; Zhen, Li; Yonghe, Zhao; Lihua, Li

    2009-01-01

    To observe the changes of adenylate cyclase (AC) and guanylate cyclase (GC) in the cerebral regions including the locus ceruleus, periaqueductal gray, and substantia nigra in rats that were physiologically dependent on morphine. We also investigated the relationship of enzymatic changes in these cerebral regions to the mechanism of morphine dependence. A morphine-dependent rat model was established and withdrawal symptoms evaluated. Enzyme histochemistry was used to detect the variations of AC and GC in cerebral regions. Compared to controls, AC and GC significantly increased in morphine-dependent groups. Comparisons of four different morphine-dependent groups also showed AC and GC significantly differed at weeks 1, 2, 4, and 8. Results found that the content of AC and GC increased in these cerebral regions in rats that demonstrated morphine dependence and appeared to be closely linked to increases in AC and GC activity.

  20. 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.

  1. Inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol in mouse spleen cells: a potential mechanism for cannabinoid-mediated immunosuppression.

    Science.gov (United States)

    Schatz, A R; Kessler, F K; Kaminski, N E

    1992-01-01

    The ability of delta 9-Tetrahydrocannabinol (delta 9-THC) to modulate adenylate cyclase activity in mouse spleen cells was investigated. These studies were prompted by the recent identification and cloning of a G-protein coupled cannabinoid receptor localized in certain regions of the brain and the potential for a common mechanism between cannabinoid-mediated CNS effects and immunosuppression. Temporal addition studies were initially performed to identify the period of time when spleen cells in culture were most susceptible to the inhibitory effects of delta 9-THC, as measured by the day 5 IgM antibody forming cell response. delta 9-THC was only inhibitory when added to spleen cell cultures during the first 2 hr following antigen sensitization. In light of this time course, adenylate cyclase activity was measured in spleen cells incubated in the presence of 22 microM delta 9-THC for 5 min and subsequently stimulated with forskolin. delta 9-THC treated spleen cells demonstrated a 33% inhibition and a 66% inhibition in intracellular cAMP after a 5 or 15 min stimulation with forskolin, respectively. These studies suggest that inhibition of immune function by delta 9-THC may be mediated through the inhibition of intracellular cAMP early after antigen stimulation.

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

    Directory of Open Access Journals (Sweden)

    Gabor Maasz

    2017-02-01

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

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

    Science.gov (United States)

    Kiss, Tibor; Jungling, Adel

    2017-01-01

    ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP. PMID:28067625

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

    Science.gov (United States)

    Maasz, Gabor; Zrinyi, Zita; Reglodi, Dora; Petrovics, Dora; Rivnyak, Adam; Kiss, Tibor; Jungling, Adel; Tamas, Andrea; Pirger, Zsolt

    2017-02-01

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

  5. Changes of Adenylate Cyclase and Guanylate Cyclase in the Frontal Cortex, Lenticula, Corpus Amygdaloideum, and Hippocampus in Morphine-dependent Rats

    Directory of Open Access Journals (Sweden)

    Shijun Hong

    2016-01-01

    Full Text Available To detect the changes of adenylate cyclase (AC and guanylate cyclase (GC in the four cerebral regions that are concerned with psychogenic dependence of morphine in rats, including the frontal cortex, lenticula, corpus amygdaloideum, and hippocampus. To discuss the relation between the expressions of AC and GC with the psychogenic dependence on morphine. Different periods of morphine-dependent rat models were established, and enzyme histochemistry was used to detect the variations of AC and GC in four cerebral regions. Compared with the control group, AC and GC in all the morphine-dependent groups increased. The data indicated that the amounts of AC and GC were significantly different between the morphine-dependent groups and the control group when tested at periods of 1 week, 2 weeks, 4 weeks, and 8 weeks (P ˂ 0.05 or P ˂ 0.01. There were significant differences when comparing the 1-week group with the 2-week, 4-week, and 8-week groups (P ˂ 0.05 or P ˂ 0.01. There were significant differences when comparing the 2-week dependent group with the 4-week dependent group or the 8-week dependent group (P ˂ 0.05 or P ˂ 0.01. The activities of AC and GC increased in four cerebral regions of morphine-dependent rats. The psychogenic dependence on morphine appears to be closely linked to the upgrade of AC and GC.

  6. 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.

  7. Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells.

    Science.gov (United States)

    Yu, Xia-Fei; Ni, Qi-Chao; Chen, Jin-Peng; Xu, Jun-Fei; Jiang, Ying; Yang, Shu-Yun; Ma, Jing; Gu, Xiao-Ling; Wang, Hua; Wang, Ying-Ying

    2014-04-01

    Adenylate cyclase-associated protein 1 (CAP1) is a conserved protein that was found to be up-regulated in breast cancer and related to the migration of breast cancer. We verified its roles in breast cancer specimens and cell lines. In our results, 71 of 100 specimens of breast cancer showed high levels of CAP1 by immunohistochemistry. Associated with statistical analysis, we saw that CAP1 was related to the grade of breast cancer. In MDA-MB-231, the expression of CAP1 was the highest and by knocking down the expression of CAP1 in MDA-MB-231, its ability for proliferating and migrating apparently decreased and induced changes in morphology, which were related to the arrangement of F-actin. Therefore, CAP1 might be a potential molecular targeted therapy for surgery and immune treatment.

  8. 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.

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

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    Liu Shuyang

    2012-08-01

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

  10. 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...

  11. High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells.

    Science.gov (United States)

    Bao, Zhen; Qiu, Xiaojun; Wang, Donglin; Ban, Na; Fan, Shaochen; Chen, Wenjuan; Sun, Jie; Xing, Weikang; Wang, Yunfeng; Cui, Gang

    2016-04-01

    Adenylate cyclase-associated protein 1 (CAP1), a conserved member of cyclase-associated proteins was reported to be associated with the proliferation, migration or invasion of the tumors of pancreas, breast and liver, and was involved in astrocyte proliferation after acute Traumatic Brain Injury (TBI). In this study, we sought to investigate the character of CAP1 in the pathological process of human glioma by detecting human glioma specimens and cell lines. 43 of 100 specimens showed high expression of CAP1 via immunohistochemistry. With statistics analysis, we found out the expression level of CAP1 was correlated with the WHO grades of human glioma and was great positively related to Ki-67 (p<0.01). In vitro, silencing CAP1 in U251 and U87MG, the glioma cell lines with the relatively higher expression of CAP1, induced the proliferation of the cells significantly retarded, migration and invasion as well. Obviously, our results indicated that CAP1 participated in the molecular pathological process of glioma indeed, and in a certain sense, CAP1 might be a potential and promising molecular target for glioma diagnosis and therapies in the future.

  12. Studies on responsiveness of hepatoma cells to catecholamines. III. Difference between the receptor-adenylate cyclase regulating systems in AH130 cells and cultured normal rat liver cells.

    Science.gov (United States)

    Sanae, F; Matsunaga, T; Miyamoto, K; Koshiura, R

    1986-10-01

    The responsiveness to three beta-adrenergic agonists, isoproterenol (IPN), epinephrine (Epi) and norepinephrine (NE) in AH13O cells was examined compared with that in normal rat liver cells which were cultured for 24 hr after collagenase digestion. As regards to the activation of adenylate cyclase in the cell homogenates, the relative affinity of the three agonists was in order of IPN greater than NE greater than Epi in AH130 cells and IPN greater than Epi greater than NE in cultured normal liver cells. While the efficacies of the three agonists were similar in cultured liver cells, those of NE and Epi were markedly lower than that of IPN in AH13O cells and were increased to the similar level of IPN by pretreatment with phentolamine, but not with prazosin. Clonidine inhibited the activation of adenylate cyclase by IPN in AH13O cells. When cells were preincubated with islet-activating protein (IAP), the activity of adenylate cyclase in the presence or absence of agonist in both cell lines increased. In IAP-treated AH13O cells, the efficacies of NE and Epi became close to that of IPN. Adenylate cyclase in IAP-treated AH13O cells was activated by GTP in a dose-dependent manner, but that in IAP-treated cultured liver cells was not. In the presence of IPN, biphasic (activatory and inhibitory) effects of GTP on the cyclase were observed, and the inhibitory phase was eliminated by the IAP-treatment in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

    Science.gov (United States)

    Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

    2013-06-01

    Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

  14. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats

    OpenAIRE

    Meloni, Edward G.; Venkataraman, Archana; Donahue, Rachel J.; Carlezon, William A.

    2015-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 ug) or vehicle followed 30 min later by fear conditioning...

  15. (/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.

  16. 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.

  17. The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity.

    Science.gov (United States)

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

    2010-05-01

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

  18. The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells.

    Science.gov (United States)

    Schlesinger, D H; Goldstein, G; Niall, H D

    1975-05-20

    The complete amino acid sequence was determined for bovine ubiquitin, and adenylate cyclase stimulating polypeptide, which is probably represented universally in living cells. Ubiquitin has a molecular weight of 8451 and consists of a single polypeptide chain containing 74 amino acid residues. It contains four arginine residues but no cysteine or trytophan residues. The first 61 amino acid residues were obtained by automated Edman degradations. Tryptic digestion of maleated ubiquitin yielded four peptide fragments that were resolved by molecular sieve chromatography and coded in order of decreasing chain length (MT-1, MT-2, MT-3, and MT-4). The automated sequenator determinations on native ubiquintin provided overlapping sequence data for three of these fragments that gave an order of MT-1, MT-3, and then MT-2; Peptide MT-4, a dipeptide, was therefore assigned to the C terminus, and the placement of peptide MT-2 was corroborated by analysis of data from carboxypeptidase digestions of maleated ubiquitin. Peptide MT-2 was domaleated and sequenced by manual Edman degradations through a single lysine residue. It was cleaved at this residue with trypsin, and the two resultant peptides were separated by ion-exchange chromatography. Manual sequencing of the C-terminal demaleated tryptic peptide of MT-2 completed the sequence of MT-2 and that of native ubiquitin. The sequence of ubiquitin was further confirmed and supported by amino acid and parital sequence anlysis of fragments obtained by digestion of maleated ubiquitin with chymotrypsin or staphylococcal protease.

  19. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

    Science.gov (United States)

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-04-24

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

  20. Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

    Science.gov (United States)

    Brown, Justin T; Kant, Andrew; Mailman, Richard B

    2009-03-15

    Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

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

    Science.gov (United States)

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

    2010-03-01

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

  2. 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.

  3. Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity and mRNA expression in the duck gastrointestinal tract.

    Science.gov (United States)

    Mirabella, N; Squillacioti, C; Colitti, M; Germano, G; Pelagalli, A; Paino, G

    2002-06-01

    The presence and distribution of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity were studied in the duck gastrointestinal tract using immunohistochemistry and radioimmunoassays. Expression and distribution of PACAP mRNA were also studied using reverse transcriptase polymerase chain reaction (RT-PCR) and hybridization techniques. In addition, a partial coding sequence (cds) of the duck growth hormone-releasing hormone (GRF)/PACAP gene was identified. The presence of both PACAP-38 and PACAP-27 was demonstrated, the former being the predominant form. PACAP immunoreactivity was found in neurons and fibers of the enteric nervous system (ENS), in endocrine cells and in the gut associated lymphoid tissue (GALT). Double immunostaining showed that PACAP is almost completely colocalized with vasoactive intestinal peptide (VIP) in the ENS. Moreover, PACAP was also found in nitric oxide synthase (NOS)-containing neurons and nerve fibers. Radioimmunoassay (RIA) performed on denervated gut showed that more than one-half of the duodenal PACAP is extrinsic in origin. RT-PCR, Northern blot analysis and in situ hybridization confirmed the immunohistochemical data. The findings of the present study suggest that, in birds, PACAP may have multiple roles in regulating gastrointestinal functions.

  4. 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.

  5. 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.

  6. 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.

  7. Augmented cystine-glutamate exchange by pituitary adenylate cyclase-activating polypeptide signaling via the VPAC1 receptor

    Science.gov (United States)

    Resch, Jon M.; Albano, Rebecca; Liu, XiaoQian; Hjelmhaug, Julie; Lobner, Doug; Baker, David A.; Choi, SuJean

    2014-01-01

    In the central nervous system, cystine import in exchange for glutamate through system xc− is critical for the production of the antioxidant glutathione by astrocytes, as well as the maintenance of extracellular glutamate. Therefore, regulation of system xc− activity affects multiple aspects of cellular physiology and may contribute to disease states. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuronally-derived peptide that has already been demonstrated to modulate multiple aspects of glutamate signaling suggesting PACAP may also target activity of cystine-glutamate exchange via system xc−. In the current study, 24-hour treatment of primary cortical cultures containing neurons and glia with PACAP concentration-dependently increased system xc− function as measured by radiolabeled cystine uptake. Furthermore, the increase in cystine uptake was completely abolished by the system xc− inhibitor, (S)-4-carboxyphenylglycine (CPG), attributing increases in cystine uptake specifically to system xc− activity. Time course and quantitative PCR results indicate that PACAP signaling may increase cystine-glutamate exchange by increasing expression of xCT, the catalytic subunit of system xc−. Furthermore, the potentiation of system xc− activity by PACAP occurs via a PKA-dependent pathway that is not mediated by the PAC1R, but rather the shared vasoactive intestinal polypeptide receptor VPAC1R. Finally, assessment of neuronal, astrocytic, and microglial-enriched cultures demonstrated that only astrocyte-enriched cultures exhibit enhanced cystine uptake following both PACAP and VIP treatment. These data introduce a novel mechanism by which both PACAP and VIP regulate system xc− activity. PMID:25066643

  8. Hepatic adenylate cyclase 3 is upregulated by Liraglutide and subsequently plays a protective role in insulin resistance and obesity.

    Science.gov (United States)

    Liang, Y; Li, Z; Liang, S; Li, Y; Yang, L; Lu, M; Gu, H F; Xia, N

    2016-01-25

    Recent studies have demonstrated that adenylate cyclase 3 (AC3) has a protective role in obesity. This gene resides at the pathway with glucagon-like peptide (GLP)-1. Liraglutide is a GLP-1 analog and has independent glucose and body weight (BW)-reducing effects. In the present study, we aimed to examine whether hepatic AC3 activity was regulated by Liraglutide and to further understand the effect of AC3 in reduction of BW and insulin resistance. The diabesity and obese mice were induced from db/db and C57BL/6 J mice, respectively, by high-fat diet. Liraglutide (0.1 mg kg(-1) per 12 h) was given to the mice twice daily for 12 weeks. C57BL/6 J mice fed with chow diet and obese or diabesity mice treated with saline were used as the controls. Hepatic AC3 gene expression at mRNA and protein levels was analyzed with real-time reverse transcription-PCR and western blot. Fasting blood glucose and serum insulin levels were measured and followed insulin resistance index (HOMA-IR) was evaluated according to the homeostasis model assessment. After administration of Liraglutide, BW and HOMA-IR in obese and diabesity mice were decreased, whereas hepatic AC3 mRNA and protein expression levels were upregulated. The AC3 gene expression was negatively correlated with BW, HOMA-IR and the area ratio of hepatic fat deposition in the liver. The present study thus provides the evidence that hepatic AC3 gene expression is upregulated by Liraglutide. The reduction of BW and improvement of insulin resistance with Liraglutide may be partially explained by AC3 activation.

  9. Impaired nocifensive behaviours and mechanical hyperalgesia, but enhanced thermal allodynia in pituitary adenylate cyclase-activating polypeptide deficient mice.

    Science.gov (United States)

    Sándor, K; Kormos, V; Botz, B; Imreh, A; Bölcskei, K; Gaszner, B; Markovics, A; Szolcsányi, J; Shintani, N; Hashimoto, H; Baba, A; Reglodi, D; Helyes, Z

    2010-10-01

    Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and its receptors (PAC1 and VPAC) have been shown in the spinal dorsal horn, dorsal root ganglia and sensory nerve terminals. Data concerning the role of PACAP in central pain transmission are controversial and we have recently published its divergent peripheral effects on nociceptive processes. The aim of the present study was to investigate acute somatic and visceral nocifensive behaviours, partial sciatic nerve ligation-evoked chronic neuropathic, as well as resiniferatoxin-induced inflammatory thermal and mechanical hyperalgesia in PACAP deficient (PACAP(-/-)) mice to elucidate its overall function in pain transmission. Neuronal activation was investigated with c-Fos immunohistochemistry. Paw lickings in the early (0-5 min) and late (20-45 min) phases of the formalin test were markedly reduced in PACAP(-/-) mice. Acetic acid-evoked abdominal contractions referring to acute visceral chemonociception was also significantly attenuated in PACAP knockout animals. In both models, the excitatory role of PACAP was supported by markedly greater c-Fos expression in the periaqueductal grey and the somatosensory cortex. In PACAP-deficient animals neuropathic mechanical hyperalgesia was absent, while c-Fos immunopositivity 20 days after the operation was significantly higher. In this chronic model, these neurons are likely to indicate the activation of secondary inhibitory pathways. Intraplantarly injected resiniferatoxin-evoked mechanical hyperalgesia involving both peripheral and central processes was decreased, but thermal allodynia mediated by only peripheral mechanisms was increased in PACAP(-/-) mice. These data clearly demonstrate an overall excitatory role of PACAP in pain transmission originating from both exteroceptive and interoceptive areas, it is also involved in central sensitization. This can be explained by the signal transduction mechanisms of its identified receptors, both PAC1 and VPAC

  10. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    Science.gov (United States)

    Duvall, Laura B; Taghert, Paul H

    2012-01-01

    The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  11. Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells.

    Science.gov (United States)

    Kasica, Natalia; Podlasz, Piotr; Sundvik, Maria; Tamas, Andrea; Reglodi, Dora; Kaleczyc, Jerzy

    2016-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.

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

    Science.gov (United States)

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

    2016-12-15

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

  13. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    Directory of Open Access Journals (Sweden)

    Laura B Duvall

    Full Text Available The neuropeptide Pigment Dispersing Factor (PDF is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+ LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  14. Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals.

    Science.gov (United States)

    Czegledi, Levente; Tamas, Andrea; Borzsei, Rita; Bagoly, Terez; Kiss, Peter; Horvath, Gabriella; Brubel, Reka; Nemeth, Jozsef; Szalontai, Balint; Szabadfi, Krisztina; Javor, Andras; Reglodi, Dora; Helyes, Zsuzsanna

    2011-05-15

    Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.

  15. 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.

  16. Effects of combinatorial treatment with pituitary adenylate cyclase activating peptide and human mesenchymal stem cells on spinal cord tissue repair.

    Directory of Open Access Journals (Sweden)

    Kuan-Min Fang

    Full Text Available The aim of this study is to understand if human mesenchymal stem cells (hMSCs and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI. To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD and peroxiredoxin-1/6 (Prx-1 and Prx-6, were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue.

  17. Overexpression of adenylate cyclase-associated protein 1 is associated with metastasis of lung cancer.

    Science.gov (United States)

    Tan, Min; Song, Xiaolian; Zhang, Guoliang; Peng, Aimei; Li, Xuan; Li, Ming; Liu, Yang; Wang, Changhui

    2013-10-01

    Lung cancer ranks first in both prevalence and mortality rates among all types of cancer. Metastasis is the main cause of treatment failure. Biomarkers are critical to early diagnosis and prediction and monitoring of progressive lesions. Several biomarkers have been identified for lung cancer but none have been routinely used clinically. The present study assessed the diagnostic and prognostic value of cyclase-associated protein 1 (CAP1) for lung cancer. CAP1 mRNA abundance and protein content were determined by real-time PCR and western blot analysis and/or immunostaining in biopsy specimens (24 neoplastic and 6 non-neoplastic) freshly collected at surgical lung resection, in 82 pathologically banked lung cancer specimens and in cultured non-invasive (95-C) and invasive (95-D) lung cancer cells. Multivariate regression analysis was performed to correlate immunoreactive CAP1 signal with cancer type and stage. In vitro cell migration was performed to determine the effect of RNA interference-mediated CAP1 gene silencing on invasiveness of 95-D cells. These analyses collectively demonstrated that: i) both CAP1 mRNA abundance and protein content were significantly higher in neoplastic compared to non-neoplastic specimens and in metastatic compared to non-metastatic specimens but not different between adenocarcinoma and squamous cell carcinoma; ii) immunoreactive CAP1 signal was significantly stronger in metastatic specimens and 95-D cells compared to non-metastatic specimens and 95-C cells; and iii) RNA interference-mediated CAP1 gene silencing adequately attenuated the invasive capacity of 95-D cells in vitro. These findings suggest that overexpression of CAP1 in lung cancer cells, particularly at the metastatic stage, may have significant clinical implications as a diagnostic/prognostic factor for lung cancer.

  18. Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

    Science.gov (United States)

    del Puerto, Ana; Díaz-Hernández, Juan-Ignacio; Tapia, Mónica; Gomez-Villafuertes, Rosa; Benitez, María José; Zhang, Jin; Miras-Portugal, María Teresa; Wandosell, Francisco; Díaz-Hernández, Miguel; Garrido, Juan José

    2012-01-01

    In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

  19. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.

    Science.gov (United States)

    Gieselmann, R; Mann, K

    1992-02-24

    A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast.

  20. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-02

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

  2. 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

  3. Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, H.; Negishi, M.; Ichikawa, A. (Kyoto Univ. (Japan))

    1990-11-01

    A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound (3H)iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.

  4. Reduced early and late phase insulin response to glucose in isolated spiny mouse (Acomys cahirinus) islets: a defective link between glycolysis and adenylate cyclase.

    Science.gov (United States)

    Nesher, R; Abramovitch, E; Cerasi, E

    1989-09-01

    The spiny mouse (Acomys cahirinus) exhibits low insulin responsiveness to glucose with a nearly absent early phase release. The alternative fuel-secretagogue glyceraldehyde (10 mmol/l) produced a maximal early insulin response in rat islets but failed to affect early response in Acomys; however, it potentiated the late insulin response in both species alike. Glucagon (1.5 mumol/l) potentiated the early insulin response to intermediate (8.3 mmol/l) glucose in rat and Acomys islets by two- and four-fold, respectively. Glucose doubled cyclic AMP levels in rat islets but no significant response was noted in Acomys islets. Isobutylmethylxanthine (0.1 mmol/l) and forskolin (25 mumol/l) caused a significant rise in islet cyclic AMP levels in both types of islets; however, neither agent restored the glucose stimulation of cyclic AMP in spiny mouse islets. Forskolin and isobutylmethylxanthine potentiated early and late phase insulin release in both species; however, neither augmented the early response in the Acomys to the degree observed in rat islets. Thus: (1) A deficient link exists in Acomys between glycolysis and subsequent signals. (2) These islets contain a glucose-insensitive adenylate cyclase. (3) The early insulin response may be potentiated by direct activation of adenylate cyclase. (4) The glucose effects on early and late phase insulin release are probably mediated by distinct pathways. (5) In the spiny mouse the signals mediating the early response are deranged to a greater extent than those activating the late phase insulin release.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-11-01

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

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    a significant dose-related relaxation on the NA-precontracted vessels. However, pre-incubation of the vessels with 10(-7) M PACAP-38, PACAP-27 and vaso active intestinal polypeptide (VIP) did not induce a general rightward shift of the NA concentration-response curves, although a tendency to inhibition......In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiments...... in the low-dose interval was observed. The peptides caused a significant, dose-dependent inhibition of both frequency and amplitude on the fallopian tube smooth muscle activity. The effects of the three peptides on longitudinally as well as transversally cut specimens were alike....

  7. X-linked recessive congenital muscle fiber hypotrophy with central nuclei: abnormalities of growth and adenylate cyclase in muscle tissue cultures.

    Science.gov (United States)

    Askanas, V; Engel, W K; Reddy, N B; Barth, P G; Bethlem, J; Krauss, D R; Hibberd, M E; Lawrence, J V; Carter, L S

    1979-10-01

    Muscle cells in cultures established from biopsy specimens of two children with an infantile-fatal form of X-linked recessive muscle fiber smallness with central nuclei showed an unusual ability to proliferate through numerous passages. Ultrastructurally, the cultured muscle fibers appeared very immature even after several weeks. The nuclei were large, the number of ribosomes was greatly increased, the myofibrils remained unstriated, and glycogen was accumulated in large lakes. The plasmalemma bound concanavalin A, alpha-bungarotoxin, and ruthenium red normally, but with tannic acid it did not show the dark binding of mature fibers. Biochemically, in the cultured muscle fibers, beta-adrenergic receptors were quantitatively normal. The level of adenylate cyclase in membranes was less than in cultured normal muscle; this defect could be responsible for impaired control mechanisms resulting in the other abnormalities observed.

  8. 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.

  9. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans.

    Science.gov (United States)

    Bahn, Y S; Sundstrom, P

    2001-05-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  10. Studies on responsiveness of hepatoma cells to catecholamines. VI. Characteristics of adrenoceptors and adenylate cyclase response in rat ascites hepatoma cells and human hepatoma cells.

    Science.gov (United States)

    Sanae, F; Kohei, K; Nomura, M; Miyamoto, K

    1992-06-01

    Alpha 1, alpha 2- and beta-Adrenoceptor densities and catecholamine responsiveness in established hepatoma cells, rat ascites hepatoma AH13, AH66, AH66F, AH109A, AH130 and AH7974 cells and human hepatocellular carcinoma HLF and HepG2 cells, were compared with those in normal rat hepatocytes and Chang liver cells. Alpha 1-Adrenoceptor densities measured by [3H]prazosin bindings were not detected in all hepatoma cell lines. Alpha 2-Adrenoceptor densities measured by [3H]clonidine bindings were also barely detected in hepatoma cell lines except for AH130 cells and HepG2 cells. Regarding beta-adrenoceptor, AH109A, AH130 and AH7974 cells had much more [125I]iodocyanopindolol binding sites than normal rat hepatocytes, although we could not detect the binding in HepG2 cells. Adenylate cyclase of normal rat hepatocyte and Chang liver cells were stimulated by beta 2-adrenergic agonist salbutamol, while the cyclase in hepatoma cells had no beta 2-adrenergic response but a beta 1-type response. These findings indicate that the characteristics of adrenergic response in hepatoma cell lines is very different from that in normal hepatocytes, suggesting a participation in the hepatocarcinogenesis and/or the autonomous proliferation of hepatoma cells.

  11. 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.

  12. 5-hydroxytryptamine release into human jejunum by cholera toxin.

    OpenAIRE

    Bearcroft, C P; Perrett, D.; Farthing, M J

    1996-01-01

    BACKGROUND: Cholera toxin produces intestinal secretion by activation of the adenylate cyclase complex. However animal studies have shown 5-hydroxytryptamine may be released after exposure to cholera toxin, and thereby contribute to the secretory state. AIM: To determine whether cholera toxin releases 5-hydroxytryptamine in human jejunum. SUBJECTS: Seven male subjects were given a subclinical dose of cholera toxin in a paired, controlled, randomised, double blind study. METHODS: A closed 10 c...

  13. cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects.

    Science.gov (United States)

    Beckert, Ulrike; Grundmann, Manuel; Wolter, Sabine; Schwede, Frank; Rehmann, Holger; Kaever, Volkhard; Kostenis, Evi; Seifert, Roland

    2014-09-05

    In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.

  14. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-25

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

  16. Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

    Science.gov (United States)

    Shibasaki, Y; Hayata-Takano, A; Hazama, K; Nakazawa, T; Shintani, N; Kasai, A; Nagayasu, K; Hashimoto, R; Tanida, M; Katayama, T; Matsuzaki, S; Yamada, K; Taniike, M; Onaka, Y; Ago, Y; Waschek, J A; Köves, K; Reglődi, D; Tamas, A; Matsuda, T; Baba, A; Hashimoto, H

    2015-06-25

    Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. 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.

  18. 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 saturated high-fat diet-induced obesity was not effective in triggering cardiac dysfunction and impair the beta-adrenergic signaling. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  19. Alzheimer amyloid beta-peptide A-beta25-35 blocks adenylate cyclase-mediated forms of hippocampal long-term potentiation.

    Science.gov (United States)

    Bisel, Blaine E; Henkins, Kristen M; Parfitt, Karen D

    2007-02-01

    Progressive memory loss and deposition of amyloid beta (Abeta) peptides throughout cortical regions are hallmarks of Alzheimer's disease (AD). Several studies in mice and rats have shown that overexpression of amyloid precursor protein (APP) or pretreatment with Abeta peptide fragments results in the inhibition of hippocampal long-term potentiation (LTP) as well as impairments in learning and memory of hippocampal-dependent tasks. For these studies we have investigated the effects of the Abeta(25-35) peptide fragment on LTP induced by adenylate cyclase stimulation followed immediately by application of Mg(++)-free aCSF ("chemLTP"). Treatment of young adult slices with the Abeta(25-35) peptide had no significant effect on basal synaptic transmission in area CA1, but treatment with the peptide for 20 min before inducing chemLTP with isoproterenol (ISO; 1 microM) or forskolin (FSK;10 microM) + Mg(++)-free aCSF resulted in complete blockade of LTP. In contrast, normal ISO-chemLTP was observed after treatment with the control peptide Abeta(35-25). The ability of the Abeta(25-35) peptide fragment to block this and other forms of synaptic plasticity may help elucidate the mechanisms underlying hippocampal deficits observed in animal models of AD and/or AD individuals.

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

    Directory of Open Access Journals (Sweden)

    Tamás Juhász

    2015-07-01

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

  1. Reduced expression of brain-derived neurotrophic factor in mice deficient for pituitary adenylate cyclase activating polypeptide type-I-receptor.

    Science.gov (United States)

    Zink, Mathias; Otto, Christiane; Zörner, Björn; Zacher, Christiane; Schütz, Günther; Henn, Fritz A; Gass, Peter

    2004-04-22

    In vitro pituitary adenylate cyclase activating polypeptide (PACAP) induces the expression of brain-derived neurotrophic factor (BDNF) via its specific receptor PAC1. Since BDNF has been implicated in learning paradigms and mice lacking functional PAC1 have deficits in hippocampus-dependent associative learning, we investigated whether PAC1 mutants show alterations in hippocampal expression of BDNF and its receptor TrkB. Semi-quantitative in situ-hybridization using exon-specific BDNF-probes revealed significantly reduced expression of the exon-III and exon-V-specific transcripts within the hippocampal CA3 region in PAC1-deficient mice. A similar trend was observed for the exon-I-specific transcript. The expression of the exon-III-specific transcript was also reduced within the dentate gyrus, while Trk B-expression did not differ between genotypes. Our data demonstrate that even in vivo PAC1-mediated signaling seems to play a pivotal role for the transcriptional regulation of BDNF.

  2. 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.

  3. Overexpression of adenylate cyclase-associated protein 1 may predict brain metastasis in non-small cell lung cancer.

    Science.gov (United States)

    Xie, Shuan-Shuan; Tan, Min; Lin, Hai-Yan; Xu, Lei; Shen, Chang-Xing; Yuan, Qing; Song, Xiao-Lian; Wang, Chang-Hui

    2015-01-01

    This study was designed to establish a biomarker risk model for predicting brain metastasis (BM) in non-small cell lung cancer (NSCLC). The model comprises 120 cases of NSCLC that were treated and followed up for 4 years. The patients were divided into the BM (n=50) and non-BM (other visceral metastasis and those without recurrence) (n=70) groups. Immunohistochemical and western blot analyses were performed in metastatic tissues of NSCLC. Multivariate regression analysis was performed to correlate the immunoreactive cyclase-associated protein 1 (CAP1) signal with BM. Survival analyses were performed by using the Kaplan-Meier method. CAP1 protein content and immunoreactivity were significantly increased in BM specimens compared to other-metastatic specimens. The survival analysis revealed that CAP1 overexpression was significantly associated with survival (P<0.05). The ROC test suggested that the area under the curve was 73.33% (P<0.001; 95% CI, 63.5-83.2%). When P=0.466, the sensitivity and specificity reached 79.5 and 67.1%, respectively. These findings suggested that CAP1 is involved in the BM of NSCLC, and that elevated levels of CAP1 expression may indicate a poor prognosis for patients with BM. The CAP1 molecular model may be useful in the prediction of the risk of BM in NSCLC.

  4. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats.

    Science.gov (United States)

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

    2016-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 μg) or vehicle followed 30 min later by fear conditioning. Freezing was measured early (1, 4, and 7 days) or following a delay (7, 10, and 13 days) after conditioning. PACAP (1.5 μg) produced a bi-phasic response in freezing behavior across test days: relative to controls, PACAP-treated rats showed a reduction in freezing when tested 1 or 7 days after fear conditioning that evolved into a significant elevation in freezing by the third test session in the early, but not delayed, group. Corticosterone (CORT) levels were significantly elevated in PACAP-treated rats following fear conditioning, but not at the time of testing (Day 1). Brain c-Fos expression revealed PACAP-dependent alterations within, as well as outside of, areas typically implicated in fear conditioning. Our findings raise the possibility that PACAP disrupts fear memory consolidation by altering synaptic plasticity within neurocircuits normally responsible for encoding fear-related cues, producing a type of dissociation or peritraumatic amnesia often seen in people early after exposure to a traumatic event. However, fear memories are retained such that repeated testing and memory reactivation (e.g., re-experiencing) causes the freezing response to emerge and persist at elevated levels. PACAP systems may represent an axis on which stress and exposure to trauma converge to promote maladaptive behavioral responses characteristic of psychiatric illnesses such as post-traumatic stress disorder (PTSD). Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways.

    Science.gov (United States)

    Taylor, Ruth D T; Madsen, Marita Grønning; Krause, Michael; Sampedro-Castañeda, Marisol; Stocker, Martin; Pedarzani, Paola

    2014-01-01

    The slow afterhyperpolarizing current (sIAHP ) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP , resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons.

  6. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    Science.gov (United States)

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC.

  7. [Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach].

    Science.gov (United States)

    Zhenlong, Cao; Jiangye, Hao; Yanfen, Zhou; Zhe, Zhang; Zhihua, Ni; Yuanxiang, Hu; Weili, Liu; Yongchao, Li; Daniel, R Storm; Runlin, Z Ma; Zhenshan, Wang

    2014-06-01

    Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybridization (SSH) approach, with MOEs from AC3(-/-) and AC3(+/+) mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on molecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3(+/+) and AC3(-/-) mice. The data indicate that kcnk3 gene is up-regulated significantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3(+/+)mice. The functions of these genes are closely related with K(+) channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.

  8. 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.

  9. Pituitary Adenylate Cyclase Activating Polypeptide, A Potential Therapeutic Agent for Diabetic Retinopathy in Rats: Focus on the Vertical Information Processing Pathway.

    Science.gov (United States)

    Szabadfi, K; Reglodi, D; Szabo, A; Szalontai, B; Valasek, A; Setalo, Gy; Kiss, P; Tamas, A; Wilhelm, M; Gabriel, R

    2016-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as retinal degenerations. Diabetic retinopathy (DR), the most common complication of diabetes, affects the microvasculature and neuronal architecture of the retina. We have proven earlier that PACAP is also protective in a rat model of DR. In this study, streptozotocin-induced DR was treated with intravitreal PACAP administration in order to further analyze the synaptic structure and proteins of PACAP-treated diabetic retinas, primarily in the vertical information processing pathway. Streptozotocin-treated Wistar rats received intravitreal PACAP injection three times into the right eye 2 weeks after the induction of diabetes. Morphological and molecular biological (qRT-PCR; Western blot) methods were used to analyze retinal synapses (ribbons, conventional) and related structures. Electron microscopic analysis revealed that retinal pigment epithelium, the ribbon synapses and other synaptic profiles suffered alterations in diabetes. However, in PACAP-treated diabetic retinas more bipolar ribbon synapses were found intact in the inner plexiform layer than in DR animals. The ribbon synapse was marked with C-terminal binding protein 2/Bassoon and formed horseshoe-shape ribbons, which were more retained in PACAP-treated diabetic retinas than in DR rats. These results are supported by molecular biological data. The selective degeneration of related structures such as bipolar and ganglion cells could be ameliorated by PACAP treatment. In summary, intravitreal administration of PACAP may have therapeutic potential in streptozotocin-induced DR through maintaining synapse integrity in the vertical pathway.

  10. 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

  11. Correlation between oocyte number and follicular fluid concentration of pituitary adenylate cyclase-activating polypeptide (PACAP) in women after superovulation treatment.

    Science.gov (United States)

    Koppan, M; Varnagy, A; Reglodi, D; Brubel, R; Nemeth, J; Tamas, A; Mark, L; Bodis, J

    2012-11-01

    Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n = 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

  12. 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.

  13. Pituitary adenylate cyclase-activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice.

    Science.gov (United States)

    Matoba, Yuko; Nonaka, Naoko; Takagi, Yoshitoki; Imamura, Eisaku; Narukawa, Masayuki; Nakamachi, Tomoya; Shioda, Seiji; Banks, William A; Nakamura, Masanori

    2016-09-01

    Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9-week-old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293-1299, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways

    Science.gov (United States)

    Taylor, Ruth DT; Madsen, Marita Grønning; Krause, Michael; Sampedro-Castañeda, Marisol; Stocker, Martin; Pedarzani, Paola

    2014-01-01

    The slow afterhyperpolarizing current (sIAHP) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc. PMID:23996525

  15. Racemic Salsolinol and its Enantiomers Act as Agonists of the μ-Opioid Receptor by Activating the Gi Protein-Adenylate Cyclase Pathway

    Science.gov (United States)

    Berríos-Cárcamo, Pablo; Quintanilla, María E.; Herrera-Marschitz, Mario; Vasiliou, Vasilis; Zapata-Torres, Gerald; Rivera-Meza, Mario

    2017-01-01

    Background: Several studies have shown that the ethanol-derived metabolite salsolinol (SAL) can activate the mesolimbic system, suggesting that SAL is the active molecule mediating the rewarding effects of ethanol. In vitro and in vivo studies suggest that SAL exerts its action on neuron excitability through a mechanism involving opioid neurotransmission. However, there is no direct pharmacologic evidence showing that SAL activates opioid receptors. Methods: The ability of racemic (R/S)-SAL, and its stereoisomers (R)-SAL and (S)-SAL, to activate the μ-opioid receptor was tested in cell-based (light-emitting) receptor assays. To further characterizing the interaction of SAL stereoisomers with the μ-opioid receptor, a molecular docking study was performed using the crystal structure of the μ-opioid receptor. Results: This study shows that SAL activates the μ-opioid receptor by the classical G protein-adenylate cyclase pathway with an half-maximal effective concentration (EC50) of 2 × 10−5 M. The agonist action of SAL was fully blocked by the μ-opioid antagonist naltrexone. The EC50 for the purified stereoisomers (R)-SAL and (S)-SAL were 6 × 10−4 M and 9 × 10−6 M respectively. It was found that the action of racemic SAL on the μ-opioid receptor did not promote the recruitment of β-arrestin. Molecular docking studies showed that the interaction of (R)- and (S)-SAL with the μ-opioid receptor is similar to that predicted for the agonist morphine. Conclusions: It is shown that (R)-SAL and (S)-SAL are agonists of the μ-opioid receptor. (S)-SAL is a more potent agonist than the (R)-SAL stereoisomer. In silico analysis predicts a morphine-like interaction between (R)- and (S)-SAL with the μ-opioid receptor. These results suggest that an opioid action of SAL or its enantiomers is involved in the rewarding effects of ethanol. PMID:28167903

  16. Multiple nickel-sensitive targets elicit cardiac arrhythmia in isolated mouse hearts after pituitary adenylate cyclase-activating polypeptide-mediated chronotropy.

    Science.gov (United States)

    Tevoufouet, Etienne E; Nembo, Erastus N; Distler, Fabian; Neumaier, Felix; Hescheler, Jürgen; Nguemo, Filomain; Schneider, Toni

    2017-03-01

    The pituitary adenylate cyclase-activating polypeptide (PACAP)-27 modulates various biological processes, from the cellular level to function specification. However, the cardiac actions of this neuropeptide are still under intense studies. Using control (+|+) and mice lacking (-|-) either R-type (Cav2.3) or T-type (Cav3.2) Ca(2+) channels, we investigated the effects of PACAP-27 on cardiac activity of spontaneously beating isolated perfused hearts. Superfusion of PACAP-27 (20nM) caused a significant increase of baseline heart frequency in Cav2.3(+|+) (156.9±10.8 to 239.4±23.4 bpm; p<0.01) and Cav2.3(-|-) (190.3±26.4 to 270.5±25.8 bpm; p<0.05) hearts. For Cav3.2, the heart rate was significantly increased in Cav3.2(-|-) (133.1±8.5 bpm to 204.6±27.9 bpm; p<0.05) compared to Cav3.2(+|+) hearts (185.7±11.2 bpm to 209.3±22.7 bpm). While the P wave duration and QTc interval were significantly increased in Cav2.3(+|+) and Cav2.3(-|-) hearts following PACAP-27 superfusion, there was no effect in Cav3.2(+|+) and Cav3.2(-|-) hearts. The positive chronotropic effects observed in the four study groups, as well as the effect on P wave duration and QTc interval were abolished in the presence of Ni(2+) (50μM) and PACAP-27 (20nM) in hearts from Cav2.3(+|+) and Cav2.3(-|-) mice. In addition to suppressing PACAP's response, Ni(2+) also induced conduction disturbances in investigated hearts. In conclusion, the most Ni(2+)-sensitive Ca(2+) channels (R- and T-type) may modulate the PACAP signaling cascade during cardiac excitation in isolated mouse hearts, albeit to a lesser extent than other Ni(2+)-sensitive targets.

  17. 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

  18. Protein-protein docking and analysis reveal that two homologous bacterial adenylyl cyclase toxins interact with calmodulin differently.

    Science.gov (United States)

    Guo, Qing; Jureller, Justin E; Warren, Julia T; Solomaha, Elena; Florián, Jan; Tang, Wei-Jen

    2008-08-29

    Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly ( approximately 200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a beta-hairpin region (amino acids 259-273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein.

  19. Adenylate cyclase stimulation and ocular hypertension inhibition by forskolin analogs%佛司可林类似物激动腺苷酸环化酶及抑制兔高眼压探讨

    Institute of Scientific and Technical Information of China (English)

    杨为民; 李新华; 陈植和; 聂玲辉; 王伯龄; 沈志强

    2001-01-01

    目的 从滇产毛喉鞘蕊花植物中提取到3个佛司可林类似物,即异佛司可林、去酰基佛司可林、1-乙酰基佛司可林,体外测定它们激动腺苷酸环化酶活性,并观察去酰基佛司可林及1-乙酰基佛司可林对水负荷兔高眼压的影响。 方法 体外测定激动腺苷酸环化酶活性用蛋白质结合放免法、气动式眼压计观察兔眼压。 结果 异佛司可林体外激动腺苷酸环化酶活性与佛司可林相当,去酰基佛司可林稍弱,1-乙酰基佛司可林无活性。1%去酰基佛司可林及1-乙酰基佛司可林滴眼液滴兔眼,能抑制水负荷造成的兔高眼压,抑制率最高分别达6.0%及10.9%,作用持续至少3h。 结论 研究表明异佛司可林及去酰基佛司可林具有体外激动腺苷酸环化酶活性的作用,而去酰基佛司可林及1-乙酰基佛司可林具有抑制水负荷兔高眼压的作用。%ObjectiveForskolin (FSK) analogs,isoforskolin (isoF),deacetylforskolin(deaF),and 1-acetylforskolin(1-aF),extracted from Coleus forskohlii native to Yunnan,were assayed for their adenylate cyclase stimulating activities in vitro and for effects of two analogs on ocular hypertension (OHT) in water-loaded rabbits.MethodsAdenylate cyclase stimulation was determined by protein-binding method of radioimmunoassay,and intraocular pressure was monitored by pneumatonometer.ResultsIt showed that isoforskolin and forskolin stimulated adenylate cyclase in vitro with almost equal activity,deacetylforskolin with milder activity,and 1-acetylforskolin with little activity in vitro.1% deaF and 1-aF suppressed rabbit OHT induced by water-loading for at least 3h,with the maximal inhibitory rates of 6.0,10.9% respectively.ConclusionThis study suggests that two foskolin analogs (isoforskolin,deacetylforskolin) possess adenylate cyclase stimulation activities in vitro;deacetylforskolin and 1

  20. Radiolabelling of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Santos, R.G.; Neves, Nicoli M.J. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Abdalla, L.F.; Brandao, R.L.; Etchehebehere, L. [Ouro Preto Univ., MG (Brazil). Escola de Farmacia. Lab. de Fisiologia e Bioquimica de Microorganismos; Lima, M.E. de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Bioquimica e Imunologia; Nicoli, J.R. [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Microbiologia

    1999-11-01

    Binding of cholera toxin to ganglioside receptors of enterocyte microvilli catalyzes the activation of adenylate cyclase causing a rise in cAMP which final result is a copious diarrhea. Saccharomyces boulardii, a nonpathogenic yeast has been used to prevent diarrhea. Although the antidiarrheic properties of S. boulardii are widely recognized, this yeast has been used on empirical basis, and the mechanism of this protective effect is unknown. The addition of cholera toxin to S. boulardii induces the raising of cAMP that triggers the activation of neutral trehalase. This suggests that toxin specifically binding to cells, is internalized and active the protein phosphorylation cascade. Our objective is labeling the cholera toxin to verify the presence of binding sites on yeast cell surfaces for the cholera toxin. Cholera toxin was radiolabelled with Na {sup 125} I by a chloramine-T method modified from Cuatrecasas and Griffiths et alii. The {sup 125} I-Cholera toxin showed a specific radioactivity at about 1000 cpm/fmol toxin. Biological activity of labeled cholera toxin measured by trehalase activation was similar to the native toxin. (author) 5 refs., 3 figs.; e-mail: nevesmj at urano.cdtn.br

  1. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis

    Science.gov (United States)

    Zhang, Xuebin; Candas, Mehmet; Griko, Natalya B.; Taussig, Ronald; Bulla, Lee A.

    2006-01-01

    Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg2+-dependent cytotoxic event upon binding of the toxin to its receptor BT-R1. Here we show that binding of Cry toxin to BT-R1 provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (Gαs) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R1 in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides. PMID:16788061

  2. Influence of N-phenyl-2-naphthylamine on the Activity of Adenylate Cyclase Signaling System and the Virulence of Clavibacter michiganensis subsp. sepedonicus

    Directory of Open Access Journals (Sweden)

    A.M. Goncharova

    2016-11-01

    Full Text Available The effect of N-phenyl-2-naphthylamine was obtained from exudates of pea root on growth, virulence and signaling-specific of potato phytopathogen Clavibacter michiganensis subsp. sepedonicus. It is shown that the compound in a physiological concentration of the peas 9 mkM had no effect on C. michiganensis subsp. sepedonicus, but when exposed to N-FNA in a concentration of 45 mkM was observed reduction in growth of planktonic culture C. michiganensis subsp. sepedonicus, as well as changes in the activity of adenylyl cyclase signaling system components in this phytopathogen.

  3. 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.

  4. 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。

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

    Science.gov (United States)

    2005-10-01

    shown that the adverse effects of the SEs and TSST-1 are naturally poten- tiated by a ubiquitous component of all gram-negative bacte- ria, namely...5). Inherent characteristics of edema toxin and other procaryotic adenylate cyclases from Bordetella pertussis, Pseudomonas aeruginosa, and Yersinia...various groups (11, 28), and this effect is linked to gene transcription (9). As evidenced with other cell types (18), the cAMP levels in human

  6. The expression and significance of adenylate cyclase-associated protein 2 in human hepatocellular carcinoma%腺苷酸环化酶相关蛋白2在肝癌组织中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    谢艳英; 徐秋霞; 韩双印; 张立达; 白阳秋; 杨玉秀

    2010-01-01

    @@ 有关腺苷酸环化酶相关蛋白(adenylate cyclase-associated protein,CAP)2在肝癌中的生物学作用机制尚不明确.本研究应用逆转录聚合酶链反应(RT-PCR)及免疫组织化学技术检测CAP2在人正常肝脏、肝硬化和肝癌组织中的表达,探讨CAP2在肝癌发生发展过程中的作用.

  7. Lack of immunological analogy between the beta-subunits of cholera toxin and human choriogonadotropin.

    Science.gov (United States)

    Acevedo, H F; Kellen, J A

    1986-01-01

    A chemical relatedness has been described between the beta-subunit of cholera toxin and that of the four dimeric glycoprotein hormones (hCG, hLH, hFSH and hTSH). However, antibodies induced by cholera toxin did not crossreact, when tested by labeled hCG binding and immunocytochemistry, with the beta-subunit of hCG. It appears that differences in the tertiary structures, as shown in this study, account for distinct epitopes. Similarities in biological activity between these two compounds, such as induction of adenyl cyclase or a protective effect against some tumors, are not based on immunological mechanisms.

  8. 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.

  9. 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.

  10. 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 ...

  11. Sensitization of spinal cord nociceptive neurons with a conjugate of substance P and cholera toxin

    Directory of Open Access Journals (Sweden)

    Perez Federico M

    2007-05-01

    Full Text Available Abstract Background Several investigators have coupled toxins to neuropeptides for the purpose of lesioning specific neurons in the central nervous system. By producing deficits in function these toxin conjugates have yielded valuable information about the role of these cells. In an effort to specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P to the catalytic subunit of cholera toxin (SP-CTA. This conjugate should be taken up selectively by neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and neuronal firing. Results The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected with either the NK1 or NK2 receptor, but not the NK3 receptor. We further demonstrate that intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB and also enhances the expression of the immediate early gene c-Fos. Behaviorally, low doses of SP-CTA (1 μg injected intrathecally produce thermal hyperalgesia. At higher doses (10 μg peripheral sensitivity is suppressed suggesting that descending inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons. Conclusion The finding that stimulation of adenylate cyclase in neurokinin receptor expressing neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore, these results demonstrate that SP-CTA can be used as a tool to study sensitization of central neurons in vivo in the absence of an injury.

  12. Channel formation by RTX-toxins of pathogenic bacteria: Basis of their biological activity.

    Science.gov (United States)

    Benz, Roland

    2016-03-01

    The pore-forming cytolysins of the RTX-toxin (Repeats in ToXin) family are a relatively small fraction of a steadily increasing family of proteins that contain several functionally important glycine-rich and aspartate containing nonapeptide repeats. These cytolysins produced by a variety of Gram-negative bacteria form ion-permeable channels in erythrocytes and other eukaryotic cells. Hemolytic and cytolytic RTX-toxins represent pathogenicity factors of the toxin-producing bacteria and are very often important key factors in pathogenesis of the bacteria. Channel formation by RTX-toxins lead to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. Here we discuss channel formation and channel properties of some of the best known RTX-toxins, such as α-hemolysin (HlyA) of Escherichia coli and the uropathogenic EHEC strains, the adenylate cyclase toxin (ACT, CyaA) of Bordetella pertussis and the RTX-toxins (ApxI, ApxII and ApxIII) produced by different strains of Actinobacillus pleuropneumoniae. The channels formed by these RTX-toxins in lipid bilayers share some common properties such as cation selectivity and voltage-dependence. Furthermore the channels are transient and show frequent switching between different ion-conducting states. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rybin, V.O.; Gureeva, A.A.

    1986-05-10

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

  14. 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.

  15. Adenylate-forming enzymes

    Science.gov (United States)

    Schmelz, Stefan; Naismith, James H.

    2012-01-01

    Thioesters, amides and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. Recently there have been a number of studies of such enzymes and in this review we suggest a new classification scheme. The review highlights the diversity in enzyme fold, active site architecture and metal coordination that has evolved to catalyze this particular reaction. PMID:19836944

  16. Pituitary adenylate cyclase activating polypeptide (PACAP), stress, and sex hormones.

    Science.gov (United States)

    King, S Bradley; Toufexis, Donna J; Hammack, Sayamwong E

    2017-06-14

    Stressor exposure is associated with the onset and severity of many psychopathologies that are more common in women than men. Moreover, the maladaptive expression and function of stress-related hormones have been implicated in these disorders. Evidence suggests that PACAP has a critical role in the stress circuits mediating stress-responding, and PACAP may interact with sex hormones to contribute to sex differences in stress-related disease. In this review, we describe the role of the PACAP/PAC1 system in stress biology, focusing on the role of stress-induced alterations in PACAP expression and signaling in the development of stress-induced behavioral change. Additionally, we present more recent data suggesting potential interactions between stress, PACAP, and circulating estradiol in pathological states, including PTSD. These studies suggest that the level of stress and circulating gonadal hormones may differentially regulate the PACAPergic system in males and females to influence anxiety-like behavior and may be one mechanism underlying the discrepancies in human psychiatric disorders.

  17. 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.

  18. 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.

  19. Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Angelika Kronhardt

    Full Text Available Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8 type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF and MAPKK protease (LF enzymatic components associated to protective antigen (PA binding subunit. The binding and translocation components anthrax protective antigen (PA(63 and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63 binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF and lethal factor (LF have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63. Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.

  20. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

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    Laakso Kati

    2007-10-01

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

  1. 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.

  2. 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.

  3. 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

    2014-01-01

    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. PMID:24742682

  4. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen

    2017-05-31

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

  5. Inhibition of Bacterial Toxin Activity by the Nuclear Stain, DRAQ5™.

    Science.gov (United States)

    Webb, Joshua N; Koufos, Evan; Brown, Angela C

    2016-08-01

    The repeats-in-toxin family of toxins includes proteins produced by Gram negative bacteria such as Escherichia coli (α-hemolysin), Bordetella pertussis (adenylate cyclase toxin), and Aggregatibacter actinomycetemcomitans (LtxA), which contribute to the pathogenesis of these organisms by killing host cells. In the case of LtxA produced by A. actinomycetemcomitans, white blood cells are targeted, allowing the bacteria to avoid clearance by the host immune system. In its association with target cells, LtxA binds to a receptor, lymphocyte function-associated antigen-1, as well as membrane lipids and cholesterol, before being internalized via a lysosomal-mediated pathway. The motivation for this project comes from our discovery that DRAQ5™, a membrane-permeable nuclear stain, prevents the internalization of LtxA in a Jurkat T cell line. We hypothesized that DRAQ5™, in crossing the plasma membrane, alters the properties of the membrane to inhibit LtxA internalization. To investigate how DRAQ5™ interacts with the lipid membrane to prevent LtxA internalization, we used studied DRAQ5™-mediated membrane changes in model membranes using a variety of techniques, including differential scanning calorimetry and fluorescence spectroscopy. Our results suggest that DRAQ5™ inhibits the activity of LtxA by decreasing the fluidity of the cellular lipid membrane, which decreases LtxA binding. These results present an interesting possible anti-virulence strategy; by altering bacterial toxin activity by modifying membrane fluidity, it may be possible to inhibit the pathogenicity of A. actinomycetemcomitans.

  6. Functional significance of the highly conserved Glu(570) in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin.

    Science.gov (United States)

    Kurehong, Chattip; Powthongchin, Busaba; Thamwiriyasati, Niramon; Angsuthanasombat, Chanan

    2011-05-01

    Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3((570-593)) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu(570), Gln(574), Glu(581), Ser(584) and Ser(585)) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu(570), which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction.

  7. Differences in purinergic amplification of osmotic cell lysis by the pore-forming RTX toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the role of pore size.

    Science.gov (United States)

    Masin, Jiri; Fiser, Radovan; Linhartova, Irena; Osicka, Radim; Bumba, Ladislav; Hewlett, Erik L; Benz, Roland; Sebo, Peter

    2013-12-01

    A large subgroup of the repeat in toxin (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae, which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis, which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity (>20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.

  8. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

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

  9. Stool C difficile toxin

    Science.gov (United States)

    ... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

  10. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion

    DEFF Research Database (Denmark)

    Sasaki, S; Watanabe, J; Ohtaki, H;

    2016-01-01

    BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required...... and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression...... and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. RESULTS: Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human...

  11. 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

    concentration was significantly lower in PAC1-/- compared with their wild-type littermates under control conditions as well as under a low- or high-salt diet and under treatment with the angiotensin-converting enzyme inhibitor ramipril, whereas no differences in plasma renin concentration between the genotypes......), because PACAP (1-27) applied in concentrations in the physiologic range (10 and 100 pmol/L) did not enhance renin release from isolated kidneys of PAC1 receptor knockout mice (PAC1-/-), whereas it stimulated renin release 1.38- and 2.5-fold in kidneys from wild-type mice. Moreover, plasma renin...... were detectable after water deprivation. These data show that PACAP acting on PAC1 receptors potently stimulates renin release, serving as a tonic enhancer of the renin system in vivo....

  12. 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.

  13. 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…

  14. 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

    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...... 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...

  15. 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…

  16. Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

    Science.gov (United States)

    Lapointe, Jason F.; Dunphy, Gary B.; Mandato, Craig A.

    2012-01-01

    Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6 nM) cause reduced hemocyte adhesion, while high levels (12–120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120 nM) and in vivo (12–120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the

  17. Hemocyte-hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit.

    Science.gov (United States)

    Lapointe, Jason F; Dunphy, Gary B; Mandato, Craig A

    2012-01-01

    Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell-bacteria adhesion and hemocyte-hemocyte adhesion (microaggregation). Hemocyte-microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte-hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6-120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2-120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2-6 nM) cause reduced hemocyte adhesion, while high levels (12-120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6-120 nM) and in vivo (12-120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting

  18. Activation of a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein during desensitization of Dictyostelium discoideum cells to chemotactic signals

    NARCIS (Netherlands)

    Snaar-Jagalska, B. Ewa; Es, Saskia van; Kesbeke, Fanja; Haastert, Peter J.M. van

    1991-01-01

    The chemoattractant cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum. Upon prolonged incubation with cAMP, cells become desensitized via two distinct processes: a decrease in the number of available cAMP-binding sites (down regulation) and modification of the receptor (pr

  19. INVOLVEMENT OF THE Ca2+-PROTEIN KINASE C AND ADENYLATE CYCLACE SIGNAL PATHWAYS IN THE ACTIVATION OF THYMOCYTES IN RESPONSE TO WHOLE-BODY IRRADIATION WITH LOW DOSE X-RAYS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To study the molecular mechanism of the stimulatory effect of low dose radiation(LDR) on T cell activation.Methods. Thymocytes from Kunming mice exposed to whole-body irradiation(WBI) with different doses of X-rays were analyzed for the changes in signal molecules of the phospholipase C-phosphatidylinositol biphosphate(PLC-IP2) and G protein-adenylate cyclase(AC) pathways.Results.It was found that[Ca2+]i increased in response to doses within 0.2 Gy which was most marked after 0.075 Gy and the increase was accentuated in the presence of Con A. The changes in CD3 and calcineurin(CN) expression of the thymocytes followed the same pattern as the alterations in [Ca2+]i after LDR. The expression of α,β1 and β2 isoforms of protein kinase C(PKC) was all up-regulated after 0.075 Gy with the increase in PKC-β1 expression being most marked. The cAMP/cGMP ratio and PKA activity of the thymocytes was lowered after low dose radiation and increased after doses above 0.5 Gy in a dose-dependent manner, thus giving rise to J-shaped dose-response curves. The Ca antagonist TMB-8 and cAMP stimulant cholera toxin suppressed the augmented thymocyte proliferation induced by LDR.Conclusion.Data presented in the present paper suggest that activation of the PLC-PIP2 signal pathway and suppression of the AC-cAMP signal pathway are involved in the stimulation of the thymocytes following WBI with low dose X-rays.

  20. Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin.

    Science.gov (United States)

    Karst, Johanna C; Barker, Robert; Devi, Usha; Swann, Marcus J; Davi, Marilyne; Roser, Stephen J; Ladant, Daniel; Chenal, Alexandre

    2012-03-16

    The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

  1. Integrative signaling networks of membrane guanylate cyclases: Biochemistry and physiology

    Directory of Open Access Journals (Sweden)

    Rameshwar K Sharma

    2016-09-01

    Full Text Available 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 TRP (Transient Receptor Potential 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.

  2. 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.

  3. 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.

  4. 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)

  5. 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...

  6. The flagellar adenylate kinases of Trypanosoma cruzi.

    Science.gov (United States)

    Camara, María de los Milagros; Bouvier, León A; Miranda, Mariana R; Pereira, Claudio A

    2015-01-01

    Adenylate kinases (ADK) are key enzymes involved in cell energy management. Trypanosomatids present the highest number of variants in a single cell in comparison with the rest of the living organisms. In this work, we characterized two flagellar ADKs from Trypanosoma cruzi, called TcADK1 and TcADK4, which are also located in the cell cytosol. Interestingly, TcADK1 presents a stage-specific expression. This variant was detected in epimastigotes cells, and was completely absent in trypomastigotes and amastigotes, while TcADK4 is present in the major life cycle stages of T. cruzi. Both variants are also regulated, in opposite ways, along the parasite growth curve suggesting that their expression depends on the intra- and extracellular conditions. Both, TcADK1 and TcADK4 present N-terminal extension that could be responsible for their subcellular localization. The presence of ADK variants in the flagellum would be critical for the provision of energy in a process of high ATP consumption such as cell motility.

  7. Mechanistic investigations on six bacterial terpene cyclases

    Directory of Open Access Journals (Sweden)

    Patrick Rabe

    2016-08-01

    Full Text Available The products obtained by incubation of farnesyl diphosphate (FPP with six purified bacterial terpene cyclases were characterised by one- and two-dimensional NMR spectroscopic methods, allowing for a full structure elucidation. The absolute configurations of four terpenes were determined based on their optical rotary powers. Incubation experiments with 13C-labelled isotopomers of FPP in buffers containing water or deuterium oxide allowed for detailed insights into the cyclisation mechanisms of the bacterial terpene cyclases.

  8. Coupled ATPase-adenylate kinase activity in ABC transporters

    Science.gov (United States)

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  9. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Xiaoying Zhou

    2012-09-01

    Full Text Available Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1(ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1(ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant

  10. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    Science.gov (United States)

    Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

    2012-09-01

    Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1(ΔAB) only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1(ΔAB) transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M

  11. Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

    DEFF Research Database (Denmark)

    Willemoës, Martin; Kilstrup, Mogens

    2005-01-01

    Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDPADP+(d)NTP. This reaction, suggested to occur by the tran......Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDPADP+(d)NTP. This reaction, suggested to occur...

  12. 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.

  13. Characterization of two unusual guanylyl cyclases from Dictyostelium

    NARCIS (Netherlands)

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

    2002-01-01

    Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell li

  14. Nucleoside triphosphate synthesis catalysed by adenylate kinase is ADP dependent

    DEFF Research Database (Denmark)

    Willemoes, Martin; Kilstrup, M.

    2005-01-01

    Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP + (d)NDP ¿ ADP + (d)NTP. This reaction, suggested to occur...

  15. 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...

  16. 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.

  17. Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres

    DEFF Research Database (Denmark)

    Møller, Morten; Fahrenkrug, Jan; Hannibal, J.

    1999-01-01

    Calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, colocalization, trigeminal ganglion, rat (Wistar)......Calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, colocalization, trigeminal ganglion, rat (Wistar)...

  18. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti–Medicago symbiosis

    OpenAIRE

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-01-01

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has trig...

  19. 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

    aura were randomly allocated to intravenous infusion of PACAP38 (10 pmol/kg/min) or vasoactive intestinal polypeptide (8 pmol/kg/min) over 20 min. We recorded incidence of migraine during and after infusion (0-24 h). Magnetic resonance angiography of selected extra- and intracranial arteries, blood...... peptides induced marked dilatation of the extracranial (P 0.05). PACAP38-induced vasodilatation was longer lasting (>2 h), whereas vasoactive intestinal polypeptide-induced dilatation was normalized after 2 h. We recorded elevated plasma PACAP38 at 1 h after...... and elevated plasma PACAP38 before onset of migraine-like attacks. PACAP38 has a much higher affinity for the PAC1 receptor and we therefore suggest that migraine induction by PACAP38 may be because of activation of the PAC1 receptor, which may be a future anti-migraine drug target....

  20. 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.

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

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    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

  2. 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

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

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    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.

  4. 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

  5. Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

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    Shima, F; Yamawaki-Kataoka, Y; Yanagihara, C; Tamada, M; Okada, T; Kariya, K; Kataoka, T

    1997-03-01

    Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-25

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

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

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    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.

  8. 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.

  9. Targeting bacterial toxins.

    Science.gov (United States)

    Ivarsson, Mattias E; Leroux, Jean-Christophe; Castagner, Bastien

    2012-04-23

    Protein toxins constitute the main virulence factors of several species of bacteria and have proven to be attractive targets for drug development. Lead candidates that target bacterial toxins range from small molecules to polymeric binders, and act at each of the multiple steps in the process of toxin-mediated pathogenicity. Despite recent and significant advances in the field, a rationally designed drug that targets toxins has yet to reach the market. This Review presents the state of the art in bacterial toxin targeted drug development with a critical consideration of achieved breakthroughs and withstanding challenges. The discussion focuses on A-B-type protein toxins secreted by four species of bacteria, namely Clostridium difficile (toxins A and B), Vibrio cholerae (cholera toxin), enterohemorrhagic Escherichia coli (Shiga toxin), and Bacillus anthracis (anthrax toxin), which are the causative agents of diseases for which treatments need to be improved. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Isoform-targeted regulation of cardiac adenylyl cyclase.

    Science.gov (United States)

    Ishikawa, Yoshihiro

    2003-01-01

    Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.

  11. Crystallization of cyclase-associated protein from Dictyostelium discoideum.

    Science.gov (United States)

    Hofmann, Andreas; Hess, Sonja; Noegel, Angelika A; Schleicher, Michael; Wlodawer, Alexander

    2002-10-01

    Cyclase-associated protein (CAP) is a conserved two-domain protein that helps to activate the catalytic activity of adenylyl cyclase in the cyclase-bound state through interaction with Ras, which binds to the cyclase in a different region. With its other domain, CAP can bind monomeric actin and therefore also carries a cytoskeletal function. The protein is thus involved in Ras/cAMP-dependent signal transduction and most likely serves as an adapter protein translocating the adenylyl cyclase complex to the actin cytoskeleton. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 71.2, b = 75.1, c = 162.9 A, have been obtained from Dictyostelium discoideum CAP carrying a C-terminal His tag. A complete native data set extending to 2.2 A resolution was collected from a single crystal using an in-house X-ray system. The asymmetric unit contains one molecule of CAP.

  12. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    Science.gov (United States)

    Makarchikov, Alexander F; Wins, Pierre; Janssen, Edwin; Wieringa, Bé; Grisar, Thierry; Bettendorff, Lucien

    2002-10-21

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

  13. GABAB and adenosine receptors mediate enhancement of the K+ current, IAHP, by reducing adenylyl cyclase activity in rat CA3 hippocampal neurons.

    Science.gov (United States)

    Gerber, U; Gähwiler, B H

    1994-11-01

    1. Gamma-aminobuturic acid-B (GABAB) and adenosine A1 receptors, which are expressed in hippocampal pyramidal cells, are linked to pertussis toxin-sensitive G-proteins known to be coupled negatively to the enzyme adenylyl cyclase. This study investigates the electrophysiological consequences of adenylyl cyclase inhibition in response to stimulation of these receptors. 2. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal slice cultures in presence of tetrodotoxin. The calcium-dependent potassium current (IAHP), which is very sensitive to intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), was used as an electrophysiological indicator of adenylyl cyclase activity. 3. Application of baclofen (10 microM), a selective agonist at GABAB receptors, or adenosine (50 microM) each resulted in a transient decrease followed by a significant enhancement in the amplitude of evoked IAHP. The initial reduction in amplitude of IAHP probably reflects inadequacies in voltage clamp of electronically distant dendritic sites, due to the shunting caused by concomitant activation of potassium conductance by baclofen/adenosine. Comparable increases in membrane conductance in response to the GABAA agonist, muscimol, caused a similar reduction in IAHP. The enhancement of IAHP is consistent with an inhibition of constitutively active adenylyl cyclase. 4. The receptor mediating the responses to adenosine was identified as belonging to the A1 subtype on the basis of its sensitivity to the selective antagonist 8-cyclopentyl-1,3-dipropylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Calcium regulation of adenylyl cyclase relevance for endocrine control.

    Science.gov (United States)

    Antoni, F A

    1997-01-01

    A fundamental process in the hormonal regulation of body functions is the conversion of the intercellular signal into an intracellular signal. The first recognized intracellular messengers mediating the actions of hormones were calcium ions (Ca(2+)) and adenosine 3':5' monophosphate (cAMP), which is synthesized from ATP by adenylyl cyclase. Recent work on the structure of adenylyl cyclases has shown that these enzymes are individually tailored molecular machines controlled by diverse Ca(2+)-dependent mechanisms. These include allosteric regulation of enzyme activity through the Ca(2+)-receptor protein calmodulin, apparently direct actions of Ca(2+)on the cyclase catalytic moiety and phosphorylation/dephosphorylation by Ca(2+)-regulated protein kinases and protein phosphatases. This article is a brief review of the recent developments in the area of cyclase control that forecast a major revival of the interest in cAMP-Ca(2+)interactions. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:7-14).

  15. Control of guanylate cyclase activity in the rod outer segment.

    Science.gov (United States)

    Pannbacker, R G

    1973-12-14

    Mammalian photoreceptors contain a guanylate cyclase which has a high specific activity and is inhibited by exposure of the rod outer segment to light. Several minutes are required for this inhibition to take effect, indicating that it is not a step in visual excitation. The activity of the enzyme is sensitive to the concentration of calcium ion in the medium, suggesting that light-induced changes in calcium distribution in the photoreceptor could control guanylate cyclase activity.

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

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    Shiva Mansouri

    Full Text Available 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.

  17. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    OpenAIRE

    María de los Milagros Cámara; Bouvier, León A.; Canepa, Gaspar E.; Mariana R Miranda; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a fe...

  18. Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

    Science.gov (United States)

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

  19. 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.

  20. 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.

  1. 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.

  2. Bacterial toxins as immunomodulators.

    Science.gov (United States)

    Donaldson, David S; Williams, Neil A

    2009-01-01

    Bacterial toxins are the causative agent at pathology in a variety of diseases. Although not always the primary target of these toxins, many have been shown to have potent immunomodulatory effects, for example, inducing immune responses to co-administered antigens and suppressing activation of immune cells. These abilities of bacterial toxins can be harnessed and used in a therapeutic manner, such as in vaccination or the treatment of autoimmune diseases. Furthermore, the ability of toxins to gain entry to cells can be used in novel bacterial toxin based immuno-therapies in order to deliver antigens into MHC Class I processing pathways. Whether the immunomodulatory properties of these toxins arose in order to enhance bacterial survival within hosts, to aid spread within the population or is pure serendipity, it is interesting to think that these same toxins potentially hold the key to preventing or treating human disease.

  3. Bacterial toxins: friends or foes?

    OpenAIRE

    Schmitt, C K; Meysick, K. C.; O'Brien, A D

    1999-01-01

    Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus alpha-toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we...

  4. Adenylate kinase locus 1 polymorphism and feto-placental development.

    Science.gov (United States)

    Fulvia, Gloria-Bottini; Antonio, Pietroiusti; Anna, Neri; Patrizia, Saccucci; Ada, Amante; Egidio, Bottini; Andrea, Magrini

    2011-12-01

    Recently our group has found that the correlation between birth weight and placental weight - an index of a balanced feto-placental unit development - is influenced by genetic factors. Since adenylate kinase locus 1 (AK₁) is a polymorphic enzyme that plays an important role in the synthesis of nucleotides required for many metabolic functions, we have investigated the possible role of its genetic variability in the correlation between birth weight and placental weight. 342 consecutive healthy newborn infants from the population of Rome (Italy) and 286 puerperae from another population from Central Italy were studied. The correlation coefficient between birth weight and placental weight is much higher in infants with low activity AK₁2-1 phenotype than in those with high activity AK₁1 phenotype. The difference between AK₁ and AK₁2-1 is well marked only in newborns with a gestational age greater than 38 weeks and it is not influenced by sex, maternal age and maternal smoking. A similar pattern is observed with maternal AK₁ phenotype. These results suggest that the difference in enzymatic activity between AK₁ phenotypes influencing the equilibrium among ATP, ADP, AMP and adenosine could have an important role in a balanced development of feto-placental unit. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Conformational dynamics of a ligand-free adenylate kinase.

    Directory of Open Access Journals (Sweden)

    Hyun Deok Song

    Full Text Available Adenylate kinase (AdK is a phosphoryl-transfer enzyme with important physiological functions. Based on a ligand-free open structure and a ligand-bound closed structure solved by crystallography, here we use molecular dynamics simulations to examine the stability and dynamics of AdK conformations in the absence of ligands. We first perform multiple simulations starting from the open or the closed structure, and observe their free evolutions during a simulation time of 100 or 200 nanoseconds. In all seven simulations starting from the open structure, AdK remained stable near the initial conformation. The eight simulations initiated from the closed structure, in contrast, exhibited large variation in the subsequent evolutions, with most (seven undergoing large-scale spontaneous conformational changes and approaching or reaching the open state. To characterize the thermodynamics of the transition, we propose and apply a new sampling method that employs a series of restrained simulations to calculate a one-dimensional free energy along a curved pathway in the high-dimensional conformational space. Our calculated free energy profile features a single minimum at the open conformation, and indicates that the closed state, with a high (∼13 kcal/mol free energy, is not metastable, consistent with the observed behaviors of the unrestrained simulations. Collectively, our simulations suggest that it is energetically unfavorable for the ligand-free AdK to access the closed conformation, and imply that ligand binding may precede the closure of the enzyme.

  6. Toxins from Bacteria

    OpenAIRE

    Henkel, James S.; Baldwin, Michael R.; Barbieri, Joseph T.

    2010-01-01

    Bacterial toxins damage the host at the site of bacterial infection or distanced from the site of infections. Bacterial toxins can be single proteins or organized as oligomeric protein complexes and are organized with distinct AB structure-function properties. The A domain encodes a catalytic activity; ADP-ribosylation of host proteins is the earliest post-translational modification determine to be performed by bacterial toxin, and now include glucosylation and proteolysis among other s. Bact...

  7. [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.

  8. 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.

  9. Chorea caused by toxins.

    Science.gov (United States)

    Miyasaki, Janis M

    2011-01-01

    Chorea is uncommonly caused by toxins. Anecdotal evidence from cases of toxin-induced chorea assists in our understanding of neurodegenerative diseases associated with chorea. Beginning in medieval Europe with ergotism and the "fire that twisted people," spanning to crack dancing in contemporary times and the coexistence of alcohol abuse with chorea, toxins may exert direct effects to enhance mesolimbic dopamine transmission or indirect effects through gamma-aminobutyric acid modulation. The following chapter will discuss toxins associated with chorea and the presumed pathophysiology underlying the movement disorders in these case series.

  10. 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.

  11. Role of glutaminyl cyclases in thyroid carcinomas.

    Science.gov (United States)

    Kehlen, Astrid; Haegele, Monique; Menge, Katja; Gans, Kathrin; Immel, Uta-Dorothee; Hoang-Vu, Cuong; Klonisch, Thomas; Demuth, Hans-Ulrich

    2013-02-01

    CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

  12. 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.

  13. 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.

  14. Computational identification of candidate nucleotide cyclases in higher plants.

    Science.gov (United States)

    Wong, Aloysius; Gehring, Chris

    2013-01-01

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

  15. A guanylyl cyclase-like gene is associated with Gibberella ear rot resistance in maize (Zea mays L.).

    Science.gov (United States)

    Yuan, J; Liakat Ali, M; Taylor, J; Liu, J; Sun, G; Liu, W; Masilimany, P; Gulati-Sakhuja, A; Pauls, K P

    2008-02-01

    Gibberella ear rot, caused by the fungal pathogen Fusarium graminearum Schwabe, is a serious disease of maize (Zea mays L.) grown in northern climates. The infected maize grain contains toxins that are very harmful to livestock and humans. A maize gene that encodes a putative 267-amino acid guanylyl cyclase-like protein (ZmGC1) was characterized and shown to be associated with resistance to this disease. The putative ZmGC1 amino acid sequence is 53% identical and 65% similar to AtGC1, an Arabidopsis guanylyl cyclase. The Zmgc1 coding sequence is nearly identical in a Gibberella ear rot-resistant line (CO387) and a susceptible line (CG62) but several nucleotide sequence differences were observed in the UTRs and introns of the two alleles. Using a 463 bp probe derived from the CG62 allele of Zmgc1 and a recombinant inbred (RI) mapping population developed from a CG62 x CO387 cross, six Zmgc1 restriction fragment length polymorphism (RFLP) fragments (ER1_1, ER1_2, ER1_3, ER1_4, ER1_5, and ER5_1) were mapped on maize chromosomes 2, 3, 7, and 8. Markers ER1_1 and ER5_1 on chromosomes 7 and 8, respectively, were significantly associated with Gibberella ear rot resistance, each in three different environments. The amount of Zmgc1 transcript in ear tissues increased more quickly and to a greater extent in the resistant genotype compared to the susceptible genotype after inoculation with F. graminearum. Zmgc1 is the first guanylyl cyclase gene characterized in maize and the first gene found to be associated with Gibberella ear rot resistance in this plant.

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

    NARCIS (Netherlands)

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

    2002-01-01

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

  17. 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.

  18. 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

  19. Protection against Shiga Toxins

    Directory of Open Access Journals (Sweden)

    Simona Kavaliauskiene

    2017-02-01

    Full Text Available Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3 on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans.

  20. Synthetic inhibitors of adenylate kinases in the assays for ATPases and phosphokinases.

    Science.gov (United States)

    Feldhau, P; Fröhlich, T; Goody, R S; Isakov, M; Schirmer, R H

    1975-09-01

    1. Procedures are given for the syntheses of alpha,omega-dinucleoside 5'-polyphosphates as inhibitors of adenylate kinases. The following order for the ability of inhibiting pig muscle adenylate kinase was observed: Ap5A greater than 1:N6-etheno-Ap5A greater than Ap6A greater than Gp5A greater than Ap4A greater than Up5A. The synthesis of adenosine tetraphosphate, the starting material for Ap5A, is also described. 2. One molecule of pig muscle adenylate kinase binds one molecule of Ap5A. The difference spectrum of Ap5A-adenylate kinase with its maximum of 5050 M-1 - cm-1 at 271 nm, as well as the fluorescence properties of 1:N6-etheno-Ap5A can be used for kinetic and binding studies. 3. The specific binding of the negatively charged Ap5A was exploited in the preparation of human muscle adenylate kinase. The enzyme was purified to homogeneity with an overall yield of 65%, the absolute value being 70 mg per kg of muscle. 4. The effect of Ap5A on adenylate kinase in extracts of various cells and cell organelles was tested. A ratio of 1:50 (mol/mol) for Ap5A to other nucleotides was used for suppressing the adenylate kinase activity in extracts of mammalian and insect skeletal muscel, of human erythrocytes and of Staphylococcus aureus. A ratio of 1:5 was found to be necessary for the adenylate kinase from tobacco leaves and spinach chloroplasts, and a ratio of 2:1 was needed for suppressing the adenylate kinase from bovine liver mitochondria, human kidney homogenate and from Escherichia coli. Ap5A appears not to be metabolized in any of the above extracts. These results indicate that Ap5A can be used for evaluating the contribution of adenylate kinase to the production of ATP fro ADP in energy-transducing systems. 5. Contaminating adenylate kinase can be inhibited by a concentration of Ap5A which does not interfere in the study of many (phospho)kinases and ATPases. The applications of Ap5A in the assay for nucleoside diphosphokinase and in the study of mechanical and

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

    OpenAIRE

    Sultana Hameeda; Neelakanta Girish; Rivero Francisco; Blau-Wasser Rosemarie; Schleicher Michael; Noegel Angelika A

    2012-01-01

    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 regul...

  2. [Role of the adenyl cyclase system in achieving the immunogenesis-stimulating action of bacterial lipopolysaccharides--pyrogenal and endogenous serum pyrogen].

    Science.gov (United States)

    Dzheksenbaev, O Sh; Selezneva, V P; Loginova, V T

    1976-05-01

    Experiments were conducted on rabbits immunized intraperitoneally with corpuscular typhoid vaccine; the number of antibody-forming cells in the spleen proved to increase after tha administration of bacterial lipopolysaccharide (LPS)--pyropeneal, and endogenous serum pyrogen (EPS) together with theopylline. The data obtained indicated that the adenylcyclase system played a certain role in the mechanism of the stimulating action of LPS and EPS.

  3. 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)

  4. 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.

  5. H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions, G protein and adenylate cyclase.

    Science.gov (United States)

    Schlicker, E; Kathmann, M; Detzner, M; Exner, H J; Göthert, M

    1994-07-01

    The present study was aimed at the identification of mechanisms following the activation of histamine H3 receptors. Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused and the (H3 receptor-mediated) effect of histamine on the electrically evoked tritium overflow was studied under a variety of conditions. The extent of inhibition produced by histamine was inversely related to the frequency of stimulation used to evoke tritium overflow and to the Ca2+ concentration in the superfusion medium. An activator (levcromakalim) and blocker (glibenclamide) of ATP-dependent K+ channels did not affect the electrically evoked tritium overflow and its inhibition by histamine. A blocker of voltage-sensitive K+ channels, tetraethylammonium (TEA), increased the evoked overflow and attenuated the inhibitory effect of histamine. TEA also reduced the inhibitory effect of noradrenaline and prostaglandin E2 on the evoked overflow. When the facilitatory effect of TEA on the evoked overflow was compensated for by reducing the Ca2+ concentration in the superfusion medium, TEA did no longer attenuate the effect of histamine. Exposure of the slices to the SH group-alkylating agent N-ethylmaleimide increased the evoked overflow and attenuated the inhibitory effect of histamine; both effects were counteracted by the SH group-protecting agent dithiothreitol, which, by itself, did not affect the evoked overflow and its inhibition by histamine. Mouse brain cortex membranes were used to study the effect of the H3 receptor agonist R-(-)-alpha-methylhistamine on the basal cAMP accumulation and on the accumulation stimulated by forskolin or noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 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...

  7. A subnanomolar concentration of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) pre-synaptically modulates glutamatergic transmission in the rat hippocampus acting through acetylcholine.

    Science.gov (United States)

    Pecoraro, Valeria; Sardone, Lara Maria; Chisari, Mariangela; Licata, Flora; Li Volsi, Guido; Perciavalle, Vincenzo; Ciranna, Lucia; Costa, Lara

    2017-01-06

    The neuropeptide PACAP modulates synaptic transmission in the hippocampus exerting multiple effects through different receptor subtypes: the underlying mechanisms have not yet been completely elucidated. The neurotransmitter acetylcholine (ACh) also exerts a well-documented modulation of hippocampal synaptic transmission and plasticity. Since PACAP was shown to stimulate ACh release in the hippocampus, we tested whether PACAP acting through ACh might indirectly modulate glutamate-mediated synaptic transmission at a pre- and/or at a post-synaptic level. Using patch clamp on rat hippocampal slices, we tested PACAP effects on stimulation-evoked AMPA receptor-mediated excitatory post-synaptic currents (EPSCsAMPA) in the CA3-CA1 synapse and on spontaneous miniature EPSCs (mEPSCs) in CA1 pyramidal neurons. A subnanomolar dose of PACAP (0.5nM) decreased EPSCsAMPA amplitude, enhanced EPSC paired-pulse facilitation (PPF) and reduced mEPSC frequency, indicating a pre-synaptic decrease of glutamate release probability: these effects were abolished by simultaneous blockade of muscarinic and nicotinic ACh receptors, indicating the involvement of endogenous ACh. The effect of subnanomolar PACAP was abolished by a PAC1 receptor antagonist but not by a VPAC receptor blocker. At a higher concentration (10nM), PACAP inhibited EPSCsAMPA: this effect persisted in the presence of ACh receptor antagonists and did not involve any change in PPF or in mEPSC frequency, thus was not mediated by ACh and was exerted post- synaptically on CA1 pyramidal neurons. We suggest that a high-affinity PAC1 receptor pre-synaptically modulates hippocampal glutamatergic transmission acting through ACh. Therefore, administration of PACAP at very low doses might be envisaged in cognitive diseases with reduced cholinergic transmission.

  8. 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

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

  9. A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase

    Science.gov (United States)

    2009-10-22

    ACKNOWLEDGMENT We thank Carol Li for technical help and Elliot Campbell for help with the stopped-flow fluorescence measurements. Dr. Ladant (Institute...387–401. 21. Ringler, P., and Schulz, G. E. (2003) Self-assembly of proteins into designed networks. Science 302, 106–109. 22. Reiersen, H., and Rees

  10. 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.

  11. 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

  12. Multilevel control of glucose homeostasis by adenylyl cyclase 8

    NARCIS (Netherlands)

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

    2015-01-01

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

  13. 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.

  14. Multilevel control of glucose homeostasis by adenylyl cyclase 8

    NARCIS (Netherlands)

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

    2015-01-01

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

  15. The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

    OpenAIRE

    Wang, J; Suzuki, N.; Kataoka, T

    1992-01-01

    In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leuci...

  16. 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.

  17. Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wins Pierre

    2008-01-01

    Full Text Available Abstract Background Thiamine triphosphate (ThTP exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. Results Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine at 37°C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. Conclusion These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis.

  18. 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

  19. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-02-18

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

  1. Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway.

    Science.gov (United States)

    Nishida, Y; Shima, F; Sen, H; Tanaka, Y; Yanagihara, C; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    1998-10-23

    In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.

  2. The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

    Science.gov (United States)

    Wang, J; Suzuki, N; Kataoka, T

    1992-11-01

    In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.

  3. Intracellular cAMP signaling by soluble adenylyl cyclase.

    Science.gov (United States)

    Tresguerres, Martin; Levin, Lonny R; Buck, Jochen

    2011-06-01

    Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cyclic adenosine 3',5' monophosphate (cAMP). sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems that are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells.

  4. Novel Structure and Function of Typhoid Toxin

    Science.gov (United States)

    ... Matters NIH Research Matters July 29, 2013 Novel Structure and Function of Typhoid Toxin Structure of typhoid toxin, showing the 2 A subunits ( ... to cultured cells. The scientists next determined the structure of the typhoid toxin. The toxin was already ...

  5. Botulinum Toxin (Botox) for Facial Wrinkles

    Science.gov (United States)

    ... Stories Español Eye Health / Eye Health A-Z Botulinum Toxin (Botox) for Facial Wrinkles Sections Botulinum Toxin (Botox) ... Facial Wrinkles How Does Botulinum Toxin (Botox) Work? Botulinum Toxin (Botox) for Facial Wrinkles Written by: Kierstan Boyd ...

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

    Science.gov (United States)

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

  7. 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.

  8. 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.

  9. NCBI nr-aa BLAST: CBRC-ACAR-01-0303 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0303 ref|NP_066939.1| brain adenylate cyclase 1 [Homo sapiens] sp|Q088...28|ADCY1_HUMAN Adenylate cyclase type 1 (Adenylate cyclase type I) (ATP pyrophosphate-lyase 1) (Adenylyl cyclase... 1) (Ca(2+)/calmodulin-activated adenylyl cyclase) gb|EAL23741.1| adenylate cyclase 1 (brain) [Homo sapiens] NP_066939.1 6e-54 57% ...

  10. [Toxins as a biological weapon].

    Science.gov (United States)

    Płusa, Tadeusz

    2015-09-01

    The criteria for recognizing a chemical compound for the toxin are vague and gave it the possibility of inclusion in this group a number of biological agents. Toxins list is extensive, but the interest is focused on bacterial toxins, poisons derived from snake venoms, algae and plant proteins, and small molecules. Particular attention is focused on the so-called "sea" toxins, which include tetrodotoxin, brevetoxin and saxitoxin. This indicates the search for a new hitherto unknown potential bioterrorist threats. © 2015 MEDPRESS.

  11. Overexpression of functional human oxidosqualene cyclase in Escherichia coli

    DEFF Research Database (Denmark)

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

    2015-01-01

    The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the te......The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation...

  12. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    Science.gov (United States)

    Bernheimer, Alan W.; Schwartz, Lois L.

    1964-01-01

    Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100–1104. 1964.—Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of β-glucuronidase and acid phosphatase. Staphylococcal α-toxin, Clostridium perfringens α-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal β-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens ε-toxin were not active in either system. Staphylococcal δ-toxin, C. histolyticum collagenase, crude C. perfringens β-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes. PMID:5874534

  13. Assessment of industrial sewage impacts by adenylate energy charge measurements in the bivalve Cerastoderma edule.

    Science.gov (United States)

    Picado, A M; Le Gal, Y

    1990-02-01

    Laboratory toxicity tests performed on the bivalve Cerastoderma edule submitted to sublethal concentrations of paper mill effluent revealed significant decreases of adenylate energy charge (AEC), and changes in the total adenylate pool were observed in a 24-hr period even for the lowest concentration of pollutant tested. Field transfer experiments of C. edule from a safe zone to polluted areas of the Sado estuary were carried out at two different times of the year. Close proximity to sewage outfall was shown to result in significant decreases in AEC values within 24 hr. One week after transfer, either normal AEC values were found or the organisms died, depending on the location of the sampling station.

  14. Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

    Science.gov (United States)

    Wang, J; Suzuki, N; Nishida, Y; Kataoka, T

    1993-07-01

    In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

  15. Dysregulation of Alternative Poly-adenylation as a Potential Player in Autism Spectrum Disorder

    Science.gov (United States)

    Szkop, Krzysztof J.; Cooke, Peter I. C.; Humphries, Joanne A.; Kalna, Viktoria; Moss, David S.; Schuster, Eugene F.; Nobeli, Irene

    2017-01-01

    We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3′ untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3′ UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3′ sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain. PMID:28955198

  16. 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.

  17. 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.

  18. 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.

  19. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    Science.gov (United States)

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  20. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    Directory of Open Access Journals (Sweden)

    Daniel Braga

    2016-12-01

    Full Text Available Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

  1. [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.

  2. 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.

  3. Autoproteolytic Activation of Bacterial Toxins

    Science.gov (United States)

    Shen, Aimee

    2010-01-01

    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. PMID:22069620

  4. Autoproteolytic activation of bacterial toxins.

    Science.gov (United States)

    Shen, Aimee

    2010-05-01

    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 (InsP(6)), 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.

  5. Altered collecting duct adenylyl cyclase content in collecting duct endothelin-1 knockout mice

    Directory of Open Access Journals (Sweden)

    Kohan Donald E

    2007-05-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 inhibition of vasopressin (AVP-stimulated water reabsorption by the inner medullary collecting duct (IMCD is associated with reduced cAMP accumulation. To determine the effect of ET-1 deficiency, AVP-stimulated cAMP responsiveness was assessed in IMCD from mice with collecting duct-specific deletion of ET-1 (CD ET-1 KO and from control animals. Methods Cyclic AMP production, adenylyl cyclase (AC mRNA, and AC protein were measured in acutely isolated IMCD. Results CD ET-1 KO IMCD had enhanced AVP-stimulated cAMP accumulation. Inhibition of calcium-stimulated AC using BAPTA did not prevent enhanced AVP responsiveness in CD ET-1 KO IMCD. Factors known to be modified by ET-1, including nitric oxide, cyclooxygenase metabolites, and superoxide did not affect the increased AVP responsiveness of CD ET-1 KO IMCD. Differential V2 receptor or G-protein activity was not involved since CD ET-1 KO IMCD had increased cAMP accumulation in response to forskolin and/or cholera toxin. CD ET-1 KO did not affect mRNA or protein levels of AC3, one of the major known collecting duct AC isoforms. However, the other known major collecting duct AC isoform (AC5/6 did have increased protein levels in CD ET-1 KO IMCD, although AC5 (weak signal and 6 mRNA levels were unchanged. Conclusion ET-1 deficiency increases IMCD AC5/6 content, an effect that may synergize with acute ET-1 inhibition of AVP-stimulated cAMP accumulation.

  6. Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.

    Science.gov (United States)

    Ramos-Espiritu, Lavoisier; Kleinboelting, Silke; Navarrete, Felipe A; Alvau, Antonio; Visconti, Pablo E; Valsecchi, Federica; Starkov, Anatoly; Manfredi, Giovanni; Buck, Hannes; Adura, Carolina; Zippin, Jonathan H; van den Heuvel, Joop; Glickman, J Fraser; Steegborn, Clemens; Levin, Lonny R; Buck, Jochen

    2016-10-01

    The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.

  7. Prokaryotic toxin-antitoxin systems: novel regulations of the toxins.

    Science.gov (United States)

    Otsuka, Yuichi

    2016-05-01

    Toxin-antitoxin (TA) systems are widely conserved in prokaryotic plasmids and chromosomes and are linked to many roles in cell physiology, including plasmid maintenance, stress response, persistence and protection from phage infection. A TA system is composed of a stable toxin and a labile antitoxin that inhibits a harmful effect of the cognate toxin. When gene expression from the TA loci is repressed under certain conditions such as nutrient starvation, the toxin is freed from the rapidly degrading antitoxin and obstructs an essential cellular process, such as DNA replication, translation and peptidoglycan synthesis, which subsequently causes growth arrest. TA systems are classified into five types according to the nature and the function of antitoxins, and the activity of toxins is tightly regulated in a variety of ways. This short-review highlights several novel regulatory mechanisms for Escherichia coli toxins that we recently discovered.

  8. Lektine, Toxine und Immunotoxine

    Science.gov (United States)

    Uhlenbruck, Gerhard

    1981-12-01

    A definition and classification of lectins (carbohydrate-binding (glyco)proteins) is given on the basis of new data and experimental results. Especially the biological role of bacterial, vertebrate and sponge lectins is discussed. The lectin-toxin combination offers an excellent model not only for studying adhesion to and penetration through the cell membrane, but also for hybridization with antibody fragments showing anti-tumor specificity.

  9. Expression of the Intestinal Biomarkers Guanylyl Cyclase C and CDX2 in Poorly Differentiated Colorectal Carcinomas

    Science.gov (United States)

    Winn, Brody; Tavares, Rosemarie; Matoso, Andres; Noble, Lelia; Fanion, Jacqueline; Waldman, Scott A.; Resnick, Murray B.

    2009-01-01

    Guanylyl cyclase C a receptor for bacterial diarrheagenic enterotoxins is expressed selectively by intestinal epithelium and is an endogenous downstream target of CDX2. The expression of Guanylyl cyclase C is preserved throughout the adenoma/carcinoma sequence in the colorectum. Detection of Guanylyl cyclase C expression by RT-PCR is currently being validated as a technique to identify occult lymph node metastases in patients with colorectal cancer and for circulating cells in the blood for postoperative surveillance. Although Guanylyl cyclase C is widely expressed by well differentiated colorectal cancer, its expression in poorly differentiated colorectal cancer has not been evaluated. A tissue microarray was created from 69 archival specimens including 44 poorly differentiated, 15 undifferentiated or medullary and 10 signet ring cell colorectal carcinomas. Matched normal colonic mucosa was used as a positive control. Immunohistochemical staining for Guanylyl cyclase C and CDX2 was evaluated as positive or negative based on at least a 10% extent of staining. Out of the 69 tumor samples 75%, 47%, and 90% of the poorly differentiated, medullary and signet ring cell tumors were positive for Guanylyl cyclase C and 75%, 40% and 90% of these subsets were positive for CDX2 respectively. There was excellent correlation between Guanylyl cyclase C and CDX2 expression on a case per case basis (p<0.0001). There was also a statistically significant difference in the GCC staining pattern between MC and PDC (p=0.05). Immunopositivity for Guanylyl cyclase C was greater than 95% in a separately stained microarray series of well/moderately differentiated colorectal carcinomas. In conclusion, Guanylyl cyclase C expression is lost in a quarter of poorly differentiated and half of undifferentiated colorectal carcinomas. Therefore the utility of Guanylyl cyclase C expression as a diagnostic marker for colorectal carcinoma may be questionable in poorly differentiated colorectal

  10. [Cytolethal distending toxins].

    Science.gov (United States)

    Curová, K; Kmeťová, M; Siegfried, L

    2014-06-01

    Cytolethal distending toxins (CDT) are intracellularly acting proteins which interfere with the eukaryotic cell cycle. They are produced by Gram-negative bacteria with affinity to mucocutaneous surfaces and could play a role in the pathogenesis of various mammalian diseases. The functional toxin is composed of three proteins: CdtB entering the nucleus and by its nuclease activity inducing nuclear fragmentation and chromatin disintegration, CdtA, and CdtC, the two latter being responsible for toxin attachment to the surface of the target cell. Cytotoxic effect of CDT leads to the cell cycle arrest before the cell enters mitosis and to further changes (cell distension and death, apoptosis) depending on the cell type. Thus, CDT may function as a virulence factor in pathogenic bacteria that produce it and thus may contribute to the initiation of certain diseases. Most important are inflammatory bowel diseases caused by intestinal bacteria, periodontitis with Aggregatibacter actinomycetemcomitans as the aetiologic agent and ulcus molle where Haemophilus ducreyi is the causative agent.

  11. Inhibition of a plant sesquiterpene cyclase by mevinolin.

    Science.gov (United States)

    Vögeli, U; Chappell, J

    1991-07-01

    The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM. Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.

  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. Intracellular trafficking of bacterial toxins.

    Science.gov (United States)

    Williams, Jeffrey M; Tsai, Billy

    2016-08-01

    Bacterial toxins often translocate across a cellular membrane to gain access into the host cytosol, modifying cellular components in order to exert their toxic effects. To accomplish this feat, these toxins traffic to a membrane penetration site where they undergo conformational changes essential to eject the toxin's catalytic subunit into the cytosol. In this brief review, we highlight recent findings that elucidate both the trafficking pathways and membrane translocation mechanisms of toxins that cross the plasma, endosomal, or endoplasmic reticulum (ER) membrane. These findings not only illuminate the specific nature of the host-toxin interactions during entry, but should also provide additional therapeutic strategies to prevent or alleviate the bacterial toxin-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death.

    Directory of Open Access Journals (Sweden)

    Ping Lin

    2016-03-01

    Full Text Available Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2 has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA. Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89 substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP.

  15. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

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

    2013-01-01

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

  16. Recombinant Toxins for Cancer Treatment

    Science.gov (United States)

    Pastan, Ira; Fitzgerald, David

    1991-11-01

    Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

  17. Intracellular Trafficking of Bacterial Toxins

    OpenAIRE

    Williams, Jeffrey M.; Tsai, Billy

    2016-01-01

    Bacterial toxins often translocate across a cellular membrane to gain access into the host cytosol, modifying cellular components in order to exert their toxic effects. To accomplish this feat, these toxins traffic to a membrane penetration site where they undergo conformational changes essential to eject the toxin’s catalytic subunit into the cytosol. In this brief review, we highlight recent findings that elucidate both the trafficking pathways and membrane translocation mechanisms of toxin...

  18. Pore formation by Cry toxins.

    Science.gov (United States)

    Soberón, Mario; Pardo, Liliana; Muñóz-Garay, Carlos; Sánchez, Jorge; Gómez, Isabel; Porta, Helena; Bravo, Alejandra

    2010-01-01

    Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

  19. NCBI nr-aa BLAST: CBRC-ACAR-01-1123 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-1123 ref|NP_899200.1| adenylate cyclase 5 [Homo sapiens] sp|O95622|ADCY5_HUMAN Adenylate cyclas...e type 5 (Adenylate cyclase type V) (ATP pyrophosphate-lyase 5) (Adenylyl cyclase 5) NP_899200.1 2e-91 52% ...

  20. THE SYNERGY OF BACTERIAL TOXINS,

    Science.gov (United States)

    TOXINS AND ANTITOXINS, STRENGTH(PHYSIOLOGY), BACTERIA, CLOSTRIDIUM PERFRINGENS, CLOSTRIDIUM TETANI , CLOSTRIDIUM , STAPHYLOCOCCUS, ESCHERICHIA COLI, PROTEUS, ETIOLOGY, ANTIGENS, ANTIBODIES, AMINO ACIDS.

  1. 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

  2. Ibogaine and noribogaine potentiate the inhibition of adenylyl cyclase activity by opioid and 5-HT receptors.

    Science.gov (United States)

    Rabin, R A; Winter, J C

    1996-12-05

    The effects of the putative anti-addictive compound ibogaine and its principal metabolite, noribogaine, on adenylyl cyclase activity were determined in various areas of the rat brain. Neither compound altered either basal or forskolin-stimulated adenylyl cyclase activities in the frontal cortex, midbrain or striatum. However, in all three brain areas the addition of ibogaine and noribogaine significantly enhanced inhibition of adenylyl cyclase activity by a maximally effective concentration of morphine. Similarly, both compounds also potentiated the inhibition of hippocampal adenylyl cyclase activity by a maximally effective concentration of 5-hydroxytryptamine (5-HT). Although ibogaine appears to be more potent than noribogaine in augmenting opioid- and 5-HT-mediated inhibition of adenylyl cyclase activity, both compounds appear to be of comparable efficacy. Neither compound, however, modified the inhibitory action of the muscarinic acetylcholine agonist, carbachol, on adenylyl cyclase activity. The present data indicate that ibogaine and noribogaine cause a selective increase in receptor-mediated inhibition of adenylyl cyclase activity. This potentiation may be involved in the pharmacological actions of these compounds.

  3. 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.

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

    Science.gov (United States)

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

    2007-07-10

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

  5. The cyclase-associated protein FgCap1 has both protein kinase A-dependent and -independent functions during deoxynivalenol production and plant infection in Fusarium graminearum.

    Science.gov (United States)

    Yin, Tao; Zhang, Qiang; Wang, Jianhua; Liu, Huiquan; Wang, Chenfang; Xu, Jin-Rong; Jiang, Cong

    2017-01-31

    Fusarium graminearum is a causal agent of wheat scab and a producer of the trichothecene mycotoxin deoxynivalenol (DON). The expression of trichothecene biosynthesis (TRI) genes and DON production are mainly regulated by the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and two pathway-specific transcription factors (TRI6 and TRI10). Interestingly, deletion mutants of TRI6 show reduced expression of several components of cAMP signalling, including the FgCAP1 adenylate-binding protein gene that has not been functionally characterized in F. graminearum. In this study, we show that FgCap1 interacts with Fac1 adenylate cyclase and that deletion of FgCAP1 reduces the intracellular cAMP level and PKA activity. The Fgcap1 deletion mutant is defective in vegetative growth, conidiogenesis and plant infection. It also shows significantly reduced DON production and TRI gene expression, which can be suppressed by exogenous cAMP, indicating a PKA-dependent regulation of DON biosynthesis by FgCap1. The wild-type, but not tri6 mutant, shows increased levels of intracellular cAMP and FgCAP1 expression under DON-producing conditions. Furthermore, the promoter of FgCAP1 contains one putative Tri6-binding site that is important for its function during DON biosynthesis, but is dispensable for hyphal growth, conidiogenesis and pathogenesis. In addition, FgCap1 shows an actin-like localization to the cortical patches at the apical region of hyphal tips. Phosphorylation of FgCap1 at S353 was identified by phosphoproteomics analysis. The S353A mutation in FgCAP1 has no effect on its functions during vegetative growth, conidiation and DON production. However, expression of the FgCAP1(S353A) allele fails to complement the defects of the Fgcap1 mutant in plant infection, indicating the importance of the phosphorylation of FgCap1 at S353 during pathogenesis. Taken together, our results suggest that FgCAP1 is involved in the regulation of DON production via cAMP signalling

  6. Effects of 39 Compounds on Calmodulin-Regulated Adenylyl Cyclases AC1 and Bacillus anthracis Edema Factor.

    Directory of Open Access Journals (Sweden)

    Carolin Lübker

    Full Text Available Adenylyl cyclases (ACs catalyze the conversion of ATP into the second messenger cAMP. Membranous AC1 (AC1 is involved in processes of memory and learning and in muscle pain. The AC toxin edema factor (EF of Bacillus anthracis is involved in the development of anthrax. Both ACs are stimulated by the eukaryotic Ca(2+-sensor calmodulin (CaM. The CaM-AC interaction could constitute a potential target to enhance or impair the AC activity of AC1 and EF to intervene in above (pathophysiological mechanisms. Thus, we analyzed the impact of 39 compounds including typical CaM-inhibitors, an anticonvulsant, an anticholinergic, antidepressants, antipsychotics and Ca(2+-antagonists on CaM-stimulated catalytic activity of AC1 and EF. Compounds were tested at 10 μM, i.e., a concentration that can be reached therapeutically for certain antidepressants and antipsychotics. Calmidazolium chloride decreased CaM-stimulated AC1 activity moderately by about 30%. In contrast, CaM-stimulated EF activity was abrogated by calmidazolium chloride and additionally decreased by chlorpromazine, felodipine, penfluridol and trifluoperazine by about 20-40%. The activity of both ACs was decreased by calmidazolium chloride in the presence and absence of CaM. Thus, CaM-stimulated AC1 activity is more insensitive to inhibition by small molecules than CaM-stimulated EF activity. Inhibition of AC1 and EF by calmidazolium chloride is largely mediated via a CaM-independent allosteric mechanism.

  7. 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.

  8. Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7*

    Science.gov (United States)

    Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H.; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

    2010-01-01

    ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca2+ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca2+ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC50 of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca2+]i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca2+]i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A. PMID:20439466

  9. Diadenosine homodinucleotide products of ADP-ribosyl cyclases behave as modulators of the purinergic receptor P2X7.

    Science.gov (United States)

    Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

    2010-07-02

    ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5',5'"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509-14514). P24, but not P18, proved to increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca(2+) through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca(2+) influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC(50) of approximately 1 mum. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca(2+)](i) has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146-23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca(2+)](i) to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.

  10. Botulinum toxin: bioweapon & magic drug.

    Science.gov (United States)

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

    2010-11-01

    Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal potency of botulinum toxin is enzymatic; the toxin is a zinc proteinase that cleaves neuronal vesicle associated proteins responsible for acetylcholine release into the neuromuscular junction. As a military or terrorist weapon, botulinum toxin could be disseminated via aerosol or by contamination of water or food supplies, causing widespread casualties. A fascinating aspect of botulinum toxin research in recent years has been development of the most potent toxin into a molecule of significant therapeutic utility . It is the first biological toxin which is licensed for treatment of human diseases. In the late 1980s, Canada approved use of the toxin to treat strabismus, in 2001 in the removal of facial wrinkles and in 2002, the FDA in the United States followed suit. The present review focuses on both warfare potential and medical uses of botulinum neurotoxin.

  11. High-sensitivity MALDI-TOF MS quantification of anthrax lethal toxin for diagnostics and evaluation of medical countermeasures.

    Science.gov (United States)

    Boyer, Anne E; Gallegos-Candela, Maribel; Quinn, Conrad P; Woolfitt, Adrian R; Brumlow, Judith O; Isbell, Katherine; Hoffmaster, Alex R; Lins, Renato C; Barr, John R

    2015-04-01

    Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics.

  12. Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

    Science.gov (United States)

    Davis, Tony D; Mohandas, Poornima; Chiriac, Maria I; Bythrow, Glennon V; Quadri, Luis E N; Tan, Derek S

    2016-11-01

    Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate β-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets.

  13. Structure of the adenylation domain of NAD(+)-dependent DNA ligase from Staphylococcus aureus.

    Science.gov (United States)

    Han, Seungil; Chang, Jeanne S; Griffor, Matt

    2009-11-01

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD(+)-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD(+)-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD(+)-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  14. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  15. High level expression of chicken muscle adenylate kinase in Escherichia coli.

    Science.gov (United States)

    Tanizawa, Y; Kishi, F; Kaneko, T; Nakazawa, A

    1987-05-01

    Chicken muscle adenylate kinase was produced in a large amount in Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the tac promoter. After induction by isopropyl-beta-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in E. coli was estimated to be 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent. Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    Science.gov (United States)

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  18. 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

  19. 2'-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa.

    Science.gov (United States)

    Das, Ushati; Shuman, Stewart

    2013-10-01

    RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3'-phosphate RNA end to a 2',3'-cyclic phosphate via covalent enzyme-(histidinyl-Nε)-AMP and RNA(3')pp(5')A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2'-phosphate RNA end in high yield. The rate of 2'-phosphate cyclization by RtcA is five orders of magnitude slower than 3'-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA3'p and RNA2'p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5'-OH ends to either 3'-phosphate or 2',3'-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2'-phosphates, which cannot be spliced by RtcB, to 2',3'-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ(54) coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.

  20. [Biosynthesis of cyclic GMP in plant cells - new insight into guanylate cyclases].

    Science.gov (United States)

    Świeżawska, Brygida; Marciniak, Katarzyna; Szmidt-Jaworska, Adriana

    2015-01-01

    Cyclic 3',5'-guanosine monophosphate (cGMP) is involved in many physiological processes in plants. Concentration of this second messenger in plant cell is determined by guanylyl cyclases (GCs) responsible for cGMP synthesis and phosphodiesterases (PDEs) involved in cGMP inactivation. First discovered plant GCs were localized in cytosol, but few years ago a new family of plasma membrane proteins with guanylyl cyclase activity was identified in Arabidopsis thaliana. These proteins belong to the family of a leucine-rich repeat receptor-like kinases (LRR-RLK) with extracellular leucine-rich repeat domain, a transmembrane-spanning domain, and an intracellular kinase domain. A novel class of guanylyl cyclases contain the GC catalytic center encapsulated within the intracellular kinase domain. These molecules are different to animal GCs in that the GC catalytic center is nested within the kinase domain. In presented paper we summarized the most recent data concerning plant guanylyl cyclases.

  1. [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.

  2. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

    OpenAIRE

    Kawamukai, M; Gerst, J; Field, J.; Riggs, M.; Rodgers, L; Wigler, M; Young, D

    1992-01-01

    We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional...

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

    OpenAIRE

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

    2012-01-01

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

  4. Choreographing the adenylyl cyclase signalosome: sorting out the partners and the steps.

    Science.gov (United States)

    Ostrom, Rennolds S; Bogard, Amy S; Gros, Robert; Feldman, Ross D

    2012-01-01

    Adenylyl cyclases are a ubiquitous family of enzymes and are critical regulators of metabolic and cardiovascular function. Multiple isoforms of the enzyme are expressed in a range of tissues. However, for many processes, the adenylyl cyclase isoforms have been thought of as essentially interchangeable, with their impact more dependent on their common actions to increase intracellular cyclic adenosine monophosphate content regardless of the isoform involved. It has long been appreciated that each subfamily of isoforms demonstrate a specific pattern of "upstream" regulation, i.e., specific patterns of ion dependence (e.g., calcium-dependence) and specific patterns of regulation by kinases (protein kinase A (PKA), protein kinase C (PKC), raf). However, more recent studies have suggested that adenylyl cyclase isoform-selective patterns of signaling are a wide-spread phenomenon. The determinants of these selective signaling patterns relate to a number of factors, including: (1) selective coupling of specific adenylyl cyclase isoforms with specific G protein-coupled receptors, (2) localization of specific adenylyl cyclase isoforms in defined structural domains (AKAP complexes, caveolin/lipid rafts), and (3) selective coupling of adenylyl cyclase isoforms with specific downstream signaling cascades important in regulation of cell growth and contractility. The importance of isoform-specific regulation has now been demonstrated both in mouse models as well as in humans. Adenylyl cyclase has not been viewed as a useful target for therapeutic regulation, given the ubiquitous expression of the enzyme and the perceived high risk of off-target effects. Understanding which isoforms of adenylyl cyclase mediate distinct cellular effects would bring new significance to the development of isoform-specific ligands to regulate discrete cellular actions.

  5. 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.

  6. 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

  7. 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).

  8. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    Science.gov (United States)

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification.

  9. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  10. Diazepam inhibits forskolin-stimulated adenylyl cyclase activity in human tumour cells.

    Science.gov (United States)

    Niles, L P; Wang, J

    1999-10-01

    Previous studies have shown that the benzodiazepine agonist, diazepam, suppresses adenylyl cyclase activity in rat brain, via a G protein-coupled benzodiazepine receptor. Since diazepam binding sites are also present in diverse non-neuronal tissues including tumour cells, its effects on adenylyl cyclase activity were examined in membranes from human MCF-7 (breast cancer) and M-6 (melanoma) cells. Diazepam caused a biphasic and concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase activity in MCF-7 membranes. The first phase of inhibition, at picomolar to nanomolar drug concentrations (EC50=5.7 x 10(-12)M), is similar to the receptor mediated phase observed in the rat brain. At micromolar concentrations of diazepam (EC50= 1.8 x 10(-4)M), the steep decrease in adenylyl cyclase activity may involve a direct action on the enzyme itself, as detected previously in rat brain membranes. Diazepam-induced suppression of adenylyl cyclase activity was also detected in M-6 membranes. However, in contrast to MCF-7 findings, only micromolar concentrations of diazepam (EC50=5.2 x 10(-4)M) inhibited enzyme activity in M-6 membranes. These findings suggest that G protein-coupled benzodiazepine receptors, which mediate inhibition of the adenylyl cyclase-cAMP pathway in the brain, are also expressed in MCF-7 cells.

  11. 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...

  12. 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.

  13. 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

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

    Science.gov (United States)

    2012-01-01

    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 polymorphic toxin systems

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

    Science.gov (United States)

    Popoff, Michel R

    2011-12-01

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

  16. The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling in Dictyostelium

    OpenAIRE

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-01-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and fo...

  17. 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.

  18. Bacterial Toxins for Cancer Therapy

    OpenAIRE

    Zahaf, Nour-Imene; Schmidt, Gudula

    2017-01-01

    Several pathogenic bacteria secrete toxins to inhibit the immune system of the infected organism. Frequently, they catalyze a covalent modification of specific proteins. Thereby, they block production and/or secretion of antibodies or cytokines. Moreover, they disable migration of macrophages and disturb the barrier function of epithelia. In most cases, these toxins are extremely effective enzymes with high specificity towards their cellular substrates, which are often central signaling molec...

  19. 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.

  20. 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.

  1. New crystal structures of adenylate kinase from Streptococcus pneumoniae D39 in two conformations.

    Science.gov (United States)

    Thach, Trung Thanh; Lee, Sangho

    2014-11-01

    Adenylate kinases (AdKs; EC 2.7.3.4) play a critical role in intercellular homeostasis by the interconversion of ATP and AMP to two ADP molecules. Crystal structures of adenylate kinase from Streptococcus pneumoniae D39 (SpAdK) have recently been determined using ligand-free and inhibitor-bound crystals belonging to space groups P21 and P1, respectively. Here, new crystal structures of SpAdK in ligand-free and inhibitor-bound states determined at 1.96 and 1.65 Å resolution, respectively, are reported. The new ligand-free crystal belonged to space group C2, with unit-cell parameters a=73.5, b=54.3, c=62.7 Å, β=118.8°. The new ligand-free structure revealed an open conformation that differed from the previously determined conformation, with an r.m.s.d on Cα atoms of 1.4 Å. The new crystal of the complex with the two-substrate-mimicking inhibitor P1,P5-bis(adenosine-5'-)pentaphosphate (Ap5A) belonged to space group P1, with unit-cell parameters a=53.9, b=62.3, c=63.0 Å, α=101.9, β=112.6, γ=89.9°. Despite belonging to the same space group as the previously reported crystal, the new Ap5A-bound crystal contains four molecules in the asymmetric unit, compared with two in the previous crystal, and shows slightly different lattice contacts. These results demonstrate that SpAdK can crystallize promiscuously in different forms and that the open structure is flexible in conformation.

  2. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    Science.gov (United States)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  3. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    Science.gov (United States)

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

    2017-01-01

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

  4. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    Science.gov (United States)

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

    2014-10-01

    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 substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

  5. 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.

  6. Targeted toxins in brain tumor therapy.

    Science.gov (United States)

    Li, Yan Michael; Hall, Walter A

    2010-11-01

    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.

  7. 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.

  8. NCBI nr-aa BLAST: CBRC-GACU-11-0020 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-11-0020 ref|NP_001107.2| adenylate cyclase 9 [Homo sapiens] sp|O60503|ADCY9_HUMAN Adenylate cyclas...e type 9 (Adenylate cyclase type IX) (ATP pyrophosphate-lyase 9) (Adenylyl cyclase ...9) gb|AAK29464.1| adenylate cyclase type 9 [Homo sapiens] gb|AAY21237.1| adenylyl cyclase type 9 [Homo sapie...ns] gb|EAW85331.1| adenylate cyclase 9, isoform CRA_a [Homo sapiens] gb|EAW85332.1| adenylate cyclase... 9, isoform CRA_a [Homo sapiens] gb|AAI51208.1| Adenylate cyclase 9 [Homo sapiens] NP_001107.2 0.0 63% ...

  9. Scorpion toxins prefer salt solutions.

    Science.gov (United States)

    Nikouee, Azadeh; Khabiri, Morteza; Cwiklik, Lukasz

    2015-11-01

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

  10. Bt toxin modification for enhanced efficacy.

    Science.gov (United States)

    Deist, Benjamin R; Rausch, Michael A; Fernandez-Luna, Maria Teresa; Adang, Michael J; Bonning, Bryony C

    2014-10-22

    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.

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

    Science.gov (United States)

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

    2012-07-31

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

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

    Directory of Open Access Journals (Sweden)

    Tian-Jun Cao

    2017-04-01

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

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

    Science.gov (United States)

    Cao, Tian-Jun; Huang, Xing-Qi; Qu, Yuan-Yuan; Zhuang, Zhong; Deng, Yin-Yin; Lu, Shan

    2017-04-11

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

  14. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    Science.gov (United States)

    Krumenacker, J. S.; Hyder, S. M.; Murad, F.

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

  15. Bacterial Toxins for Cancer Therapy.

    Science.gov (United States)

    Zahaf, Nour-Imene; Schmidt, Gudula

    2017-07-28

    Several pathogenic bacteria secrete toxins to inhibit the immune system of the infected organism. Frequently, they catalyze a covalent modification of specific proteins. Thereby, they block production and/or secretion of antibodies or cytokines. Moreover, they disable migration of macrophages and disturb the barrier function of epithelia. In most cases, these toxins are extremely effective enzymes with high specificity towards their cellular substrates, which are often central signaling molecules. Moreover, they encompass the capacity to enter mammalian cells and to modify their substrates in the cytosol. A few molecules, at least of some toxins, are sufficient to change the cellular morphology and function of a cell or even kill a cell. Since many of those toxins are well studied concerning molecular mechanisms, cellular receptors, uptake routes, and structures, they are now widely used to analyze or to influence specific signaling pathways of mammalian cells. Here, we review the development of immunotoxins and targeted toxins for the treatment of a disease that is still hard to treat: cancer.

  16. 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.

  17. Entry of Shiga toxin into cells

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; van Deurs, Bo

    1994-01-01

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

  18. Inhibition of cholera toxin and other AB toxins by polyphenolic compounds

    Science.gov (United States)

    All AB-type protein toxins have intracellular targets despite an initial extracellular location. These toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against AB toxins are therefore hard to develop because the toxins use dif...

  19. Structure of the adenylation-peptidyl carrier protein didomain of the Microcystis aeruginosa microcystin synthetase McyG.

    Science.gov (United States)

    Tan, Xiao-Feng; Dai, Ya-Nan; Zhou, Kang; Jiang, Yong-Liang; Ren, Yan-Min; Chen, Yuxing; Zhou, Cong-Zhao

    2015-04-01

    Microcystins, which are the most common cause of hepatotoxicity associated with cyanobacterial water blooms, are assembled in vivo on a large multienzyme complex via a mixed nonribosomal peptide synthetase/polyketide synthetase (NRPS/PKS). The biosynthesis of microcystin in Microcystis aeruginosa PCC 7806 starts with the enzyme McyG, which contains an adenylation-peptidyl carrier protein (A-PCP) didomain for loading the starter unit to assemble the side chain of an Adda residue. However, the catalytic mechanism remains unclear. Here, the 2.45 Å resolution crystal structure of the McyG A-PCP didomain complexed with the catalytic intermediate L-phenylalanyl-adenylate (L-Phe-AMP) is reported. Each asymmetric unit contains two protein molecules, one of which consists of the A-PCP didomain and the other of which comprises only the A domain. Structural analyses suggest that Val227 is likely to be critical for the selection of hydrophobic substrates. Moreover, two distinct interfaces demonstrating variable crosstalk between the PCP domain and the A domain were observed. A catalytic cycle for the adenylation and peptide transfer of the A-PCP didomain is proposed.

  20. 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.

  1. Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions.

    Science.gov (United States)

    Koch, K W; Stryer, L

    1988-07-07

    Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.

  2. 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...

  3. Food-Borne Bacterial Toxins

    Science.gov (United States)

    Thatcher, F. S.

    1966-01-01

    Food poisoning caused by the ingestion of preformed bacterial toxins is considered in relation to comparative symptoms, procedures for extraction and purification of the causal toxins, their chemistry, serology, assay procedures and pharmacology, in so far as these are known. The bacteria discussed in this context are Clostridium botulinum, C. perfringens, Staphylococcus aureus, Bacillus cereus, and Vibrio parahemolyticus. The possible roles of the enterococci, Proteus, E. coli and of unknown species, in relation to production of non-antigenic toxic substances, are discussed briefly. Requirements for prevention of the various forms of bacterial food poisoning are outlined. PMID:5905949

  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. 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.

  6. Multifunctional oxidosqualene cyclases and cytochrome P450involved in the biosynthesis of apple fruit triterpenic acids

    OpenAIRE

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

    2016-01-01

    Summary Apple (Malus × domestica) accumulates bioactive ursane‐, oleanane‐, and lupane‐type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology‐based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with co...

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

    OpenAIRE

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

    2007-01-01

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

  8. Binding of cholera toxin to Giardia lamblia.

    OpenAIRE

    McCardell, B. A.; Madden, J M; Stanfield, J T; Tall, B D; Stephens, M. J.

    1987-01-01

    Binding of cholera toxin to Giardia lamblia was demonstrated by two slightly different methods: an immunofluorescence technique using antibody to cholera toxin and anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate, and a one-step fluorescence method in which G. lamblia was incubated with the B subunit of cholera toxin conjugated to fluorescein isothiocyanate.

  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. Multiple lineage specific expansions within the guanylyl cyclase gene family

    Directory of Open Access Journals (Sweden)

    O'Halloran Damien M

    2006-03-01

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

  11. 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.

  12. 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.

  13. Influence of sampling, storage, processing and optimal experimental conditions on adenylate energy charge in penaeid shrimp

    Directory of Open Access Journals (Sweden)

    Robles-Romo Arlett

    2014-01-01

    Full Text Available Adenylate energy charge (AEC has been used as a practical index of the physiological status and health in several disciplines, such as ecotoxicology and aquaculture. This study standardizes several procedures for AEC determination in penaeid shrimp that are very sensitive to sampling. We concluded that shrimp can be frozen in liquid nitrogen and then stored at -76°C for up to two years for further analysis, or freshly dissected and immediately homogenized in acid. Other cooling procedures, such as immersion in cold water or placing shrimp on ice for 15 min resulted in 50% and 73% decreases in ATP levels, and 9-fold and 10-fold increases in IMP levels, respectively. Optimal values of AEC (0.9 were obtained in shrimp recently transferred from ponds to indoor conditions, but decreased to 0.77 after one month in indoor tanks when stocked at high densities; the AEC re-established to 0.85 when the shrimps were transferred to optimal conditions (lower density and dark tanks. While the levels of arginine phosphate followed the same pattern, its levels did not fully re-establish. Comparison of different devices for sample homogenization indicated that a cryogenic ball mill mixer is the more suitable procedure.

  14. 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.

  15. The energy profiles of atomic conformational transition intermediates of adenylate kinase.

    Science.gov (United States)

    Feng, Yaping; Yang, Lei; Kloczkowski, Andrzej; Jernigan, Robert L

    2009-11-15

    The elastic network interpolation (ENI) (Kim et al., Biophys J 2002;83:1620-1630) is a computationally efficient and physically realistic method to generate conformational transition intermediates between two forms of a given protein. However it can be asked whether these calculated conformations provide good representatives for these intermediates. In this study, we use ENI to generate conformational transition intermediates between the open form and the closed form of adenylate kinase (AK). Based on C(alpha)-only intermediates, we construct atomic intermediates by grafting all the atoms of known AK structures onto the C(alpha) atoms and then perform CHARMM energy minimization to remove steric conflicts and optimize these intermediate structures. We compare the energy profiles for all intermediates from both the CHARMM force-field and from knowledge-based energy functions. We find that the CHARMM energies can successfully capture the two energy minima representing the open AK and closed AK forms, while the energies computed from the knowledge-based energy functions can detect the local energy minimum representing the closed AK form and show some general features of the transition pathway with a somewhat similar energy profile as the CHARMM energies. The combinatorial extension structural alignment (Shindyalov et al., 1998;11:739-747) and the k-means clustering algorithm are then used to show that known PDB structures closely resemble computed intermediates along the transition pathway.

  16. On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Brokaw, Jason B.; Chu, Jhih-wei

    2010-11-17

    We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier works of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro177 on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro177 hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.

  17. 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.

  18. Riboswitch control of induction of aminoglycoside resistance acetyl and adenyl-transferases.

    Science.gov (United States)

    He, Weizhi; Zhang, Xuhui; Zhang, Jun; Jia, Xu; Zhang, Jing; Sun, Wenxia; Jiang, Hengyi; Chen, Dongrong; Murchie, Alastair I H

    2013-08-01

    The acquisition of antibiotic resistance by human pathogens poses a significant threat to public health. The mechanisms that control the proliferation and expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are a historically important class of antibiotics that were introduced in the 1940s. Aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug or enzymatic modification of the target rRNA through methylation or through the overexpression of efflux pumps. In our recent paper, we reported that expression of the aminoglycoside resistance genes encoding the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes was controlled by an aminoglycoside-sensing riboswitch RNA. This riboswitch is embedded in the leader RNA of the aac/aad genes and is associated with the integron cassette system. The leader RNA can sense and bind specific aminoglycosides such that the binding causes a structural transition in the leader RNA, which leads to the induction of aminoglycoside antibiotic resistance. Specific aminoglycosides induce reporter gene expression mediated by the leader RNA. Aminoglycoside RNA binding was measured directly and, aminoglycoside-induced changes in RNA structure monitored by chemical probing. UV cross-linking and mutational analysis identified potential aminoglycoside binding sites on the RNA.

  19. 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.

  20. 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.

  1. Food irradiation and bacterial toxins

    Energy Technology Data Exchange (ETDEWEB)

    Tranter, H.S.; Modi, N.K.; Hambleton, P.; Melling, J.; Rose, S.; Stringer, M.F.

    1987-07-04

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

  2. Polymer antidotes for toxin sequestration.

    Science.gov (United States)

    Weisman, Adam; Chou, Beverly; O'Brien, Jeffrey; Shea, Kenneth J

    2015-08-01

    Toxins delivered by envenomation, secreted by microorganisms, or unintentionally ingested can pose an immediate threat to life. Rapid intervention coupled with the appropriate antidote is required to mitigate the threat. Many antidotes are biological products and their cost, methods of production, potential for eliciting immunogenic responses, the time needed to generate them, and stability issues contribute to their limited availability and effectiveness. These factors exacerbate a world-wide challenge for providing treatment. In this review we evaluate a number of polymer constructs that may serve as alternative antidotes. The range of toxins investigated includes those from sources such as plants, animals and bacteria. The development of polymeric heavy metal sequestrants for use as antidotes to heavy metal poisoning faces similar challenges, thus recent findings in this area have also been included. Two general strategies have emerged for the development of polymeric antidotes. In one, the polymer acts as a scaffold for the presentation of ligands with a known affinity for the toxin. A second strategy is to generate polymers with an intrinsic affinity, and in some cases selectivity, to a range of toxins. Importantly, in vivo efficacy has been demonstrated for each of these strategies, which suggests that these approaches hold promise as an alternative to biological or small molecule based treatments.

  3. 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.

  4. 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.

  5. Antibody microarrays for native toxin detection.

    Science.gov (United States)

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E

    2005-04-15

    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  6. 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.

  7. Impairment of adenylyl cyclase signal transduction in mecobalamin-deficient rats.

    Science.gov (United States)

    Hatta, S; Watanabe, M; Ikeda, H; Kamada, H; Saito, T; Ohshika, H

    1995-11-30

    This study examined alterations in the beta-adrenoceptor-G5-adenylyl cyclase system in cerebral cortex membranes from vitamin B12-deficient rats fed a diet lacking vitamin B12 (mecobalamin) for 15 weeks. Basal, 5(7)-guanylylimidodiphosphate (GppNHp)-, isoproterenol-, and forskolin-stimulated adenylyl cyclase activities were significantly reduced in mecobalamin-deficient rats compared with those in control rats. However, no significant differences were observed in the amount and function of G5- estimated by immunoblotting and guanine nucleotide photoaffinity labeling, respectively, or in the densities and the dissociation constants of beta-adrenoceptors, estimated by [125I] pindolol binding, between control and the deficient rats. These results indicate that vitamin B12 deficiency results in the impairment of the coupling among the beta-adrenoceptor, G5- and the catalytic subunit of adenylyl cyclase, and in dysfunction of the catalytic subunit of the enzyme, suggesting that vitamin B12 participates in the regulation of neuronal adenylyl cyclase signal transduction.

  8. A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression

    NARCIS (Netherlands)

    Otsuka, Hideshi; Haastert, Peter J.M. van

    1998-01-01

    Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a muta

  9. A short history of cGMP, guanylyl cyclases, and cGMP-dependent protein kinases.

    Science.gov (United States)

    Kots, Alexander Y; Martin, Emil; Sharina, Iraida G; Murad, Ferid

    2009-01-01

    Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules.

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

    DEFF Research Database (Denmark)

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

    2015-01-01

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

  11. Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins.

    Science.gov (United States)

    Yu, G; Swiston, J; Young, D

    1994-06-01

    We previously reported the identification of human CAP, a protein that is related to the Saccharomyces cerevisiae and Schizosaccharomyces pombe adenylyl cyclase-associated CAP proteins. The two yeast CAP proteins have similar functions: the N-terminal domains are required for the normal function of adenylyl cyclase, while loss of the C-terminal domains result in morphological and nutritional defects that are unrelated to the cAMP pathways. We have amplified and cloned cDNAs from a human glioblastoma library that encode a second CAP-related protein, CAP2. The human CAP and CAP2 proteins are 64% identical. Expression of either human CAP or CAP2 in S. cerevisiae cap- strains suppresses phenotypes associated with deletion of the C-terminal domain of CAP, but does not restore hyper-activation of adenylyl cyclase by RAS2val19. Similarly, expression of either human CAP or CAP2 in S. pombe cap- strains suppresses the morphological and temperature-sensitive phenotypes associated with deletion of the C-terminal domain of CAP in this yeast. In addition, expression of human CAP, but not CAP2, suppresses the propensity to sporulate due to deletion of the N-terminal domain of CAP in S. pombe. This latter observation suggests that human CAP restores normal adenylyl cyclase activity in S. pombe cap- cells. Thus, functional properties of both N-terminal and C-terminal domains are conserved between the human and S. pombe CAP proteins.

  12. Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

    Science.gov (United States)

    Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

    2017-06-01

    Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

  13. Differential calcium signaling by cone specific guanylate cyclase-activating proteins from the zebrafish retina.

    Directory of Open Access Journals (Sweden)

    Alexander Scholten

    Full Text Available Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca(2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca(2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca(2+ around 30 nM (zGCAP1, 2 and 3 or around 400 nM (zGCAP4, 5 and 7. Zebrafish GCAP isoforms showed also differences in their Ca(2+/Mg(2+-dependent conformational changes and in the Ca(2+-dependent monomer-dimer equilibrium. Direct Ca(2+-binding revealed that all zGCAPs bound at least three Ca(2+. The corresponding apparent affinity constants reflect binding of Ca(2+ with high (≤ 100 nM, medium (0.1-5 µM and/or low (≥ 5 µM affinity, but were unique for each zGCAP isoform. Our data indicate a Ca(2+-sensor system in zebrafish rod and cone cells supporting a Ca(2+-relay model of differential zGCAP operation in these cells.

  14. A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression

    NARCIS (Netherlands)

    Otsuka, Hideshi; Haastert, Peter J.M. van

    1998-01-01

    Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a muta

  15. Nemertean toxin genes revealed through transcriptome sequencing.

    Science.gov (United States)

    Whelan, Nathan V; Kocot, Kevin M; Santos, Scott R; Halanych, Kenneth M

    2014-11-27

    Nemerteans are one of few animal groups that have evolved the ability to utilize toxins for both defense and subduing prey, but little is known about specific nemertean toxins. In particular, no study has identified specific toxin genes even though peptide toxins are known from some nemertean species. Information about toxin genes is needed to better understand evolution of toxins across animals and possibly provide novel targets for pharmaceutical and industrial applications. We sequenced and annotated transcriptomes of two free-living and one commensal nemertean and annotated an additional six publicly available nemertean transcriptomes to identify putative toxin genes. Approximately 63-74% of predicted open reading frames in each transcriptome were annotated with gene names, and all species had similar percentages of transcripts annotated with each higher-level GO term. Every nemertean analyzed possessed genes with high sequence similarities to known animal toxins including those from stonefish, cephalopods, and sea anemones. One toxin-like gene found in all nemerteans analyzed had high sequence similarity to Plancitoxin-1, a DNase II hepatotoxin that may function well at low pH, which suggests that the acidic body walls of some nemerteans could work to enhance the efficacy of protein toxins. The highest number of toxin-like genes found in any one species was seven and the lowest was three. The diversity of toxin-like nemertean genes found here is greater than previously documented, and these animals are likely an ideal system for exploring toxin evolution and industrial applications of toxins. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. 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.

  17. 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.

  18. Toxins-antitoxins: diversity, evolution and function.

    Science.gov (United States)

    Hayes, Finbarr; Van Melderen, Laurence

    2011-10-01

    Genes for toxin-antitoxin (TA) complexes are widespread in prokaryote genomes, and species frequently possess tens of plasmid and chromosomal TA loci. The complexes are categorized into three types based on genetic organization and mode of action. The toxins universally are proteins directed against specific intracellular targets, whereas the antitoxins are either proteins or small RNAs that neutralize the toxin or inhibit toxin synthesis. Within the three types of complex, there has been extensive evolutionary shuffling of toxin and antitoxin genes leading to considerable diversity in TA combinations. The intracellular targets of the protein toxins similarly are varied. Numerous toxins, many of which are sequence-specific endoribonucleases, dampen protein synthesis levels in response to a range of stress and nutritional stimuli. Key resources are conserved as a result ensuring the survival of individual cells and therefore the bacterial population. The toxin effects generally are transient and reversible permitting a set of dynamic, tunable responses that reflect environmental conditions. Moreover, by harboring multiple toxins that intercede in protein synthesis in response to different physiological cues, bacteria potentially sense an assortment of metabolic perturbations that are channeled through different TA complexes. Other toxins interfere with the action of topoisomersases, cell wall assembly, or cytoskeletal structures. TAs also play important roles in bacterial persistence, biofilm formation and multidrug tolerance, and have considerable potential both as new components of the genetic toolbox and as targets for novel antibacterial drugs.

  19. 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.

  20. Botulinum Toxin in Pediatric Neurology

    Directory of Open Access Journals (Sweden)

    Eman M. I. Moawad MD

    2015-06-01

    Full Text Available 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 pain syndromes. The use of botulinum toxin in children separate from adults has received very little attention in the literature. This review presents the current data on the use of botulinum neurotoxin to treat various neurological disorders in children.

  1. Bioinformatics Analysis of Adenylate Cyclase (AC)in Col letotrichum graminicola%禾谷炭疽菌腺苷酸环化酶的生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    韩长志

    2014-01-01

    The typical AC sequence was compared with on-line C.protein databank by Blastp comparison,then the bioinformatics of conserved domain,physicochemical property,cell signal peptide, structure of transmembrane domain and secondary structure was analyzed by SMART and the genetic relationships between typical AC sequence in C.graminicola and the homologous sequence of other species were compared to provide a reference for development and utilization of new medicines with drug target of C.graminicola.The results showed that CgCap1in C.graminicola is a homologous protein with Srv2 in S. cerevisiae.CgCap 1 located in the plasma membrane with conserved CARP domain and isoeletric point with 5~6 range belongs to the sequence of hydrophily,instable protein and without signal peptide.The proportion of βfold is low in the secondary structure.The genetic relationship between CgCap 1 and Colletoreichum is close.%为了给禾谷炭疽菌新靶标药物的研究开发提供参考,以酿酒酵母的典型腺苷酸环化酶(AC)序列为基础,利用炭疽菌属蛋白数据库在线对其进行 Blastp 比对及关键词搜索,通过 SMART 对该菌典型AC 蛋白的保守结构域、理化性质、细胞信号肽、跨膜区结构以及二级结构等生物信息学进行分析,同时比较禾谷炭疽菌中典型 AC 氨基酸序列与其他物种同源序列的遗传关系。结果表明:禾谷炭疽菌中存在一个与酿酒酵母 Srv2同源的序列,命名为 CgCap1腺苷酸环化酶蛋白,该酶蛋白包含有530个氨基酸,基因序列长1906 bp 具有保守 CARP 结构域,等电点蛋白在5~6范围,属亲水性、不稳定蛋白,不含信号肽序列,定位于质膜上,二级结构中的β折叠所占比例较低,与同属于炭疽菌属真菌胶孢炭疽菌、西瓜炭疽菌的亲缘关系较近。

  2. 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.

  3. 希金斯炭疽菌腺苷酸环化酶相关蛋白生物信息学分析%Bioinformatics analysis of adenylate cyclase associated protein in Colletotrichum higginsianum

    Institute of Scientific and Technical Information of China (English)

    韩长志

    2014-01-01

    [目的]希金斯炭疽菌可引起菜心、萝卜等十字花科植物的炭疽病,给经济产生巨大损失.目前,炭疽病菌对苯并咪唑类杀菌剂所产生的抗性问题引起了学者广泛关注,有待于开发新的作用靶标的化学药剂.腺苷酸环化酶(AC)在G蛋白信号传导途径上发挥着重要作用.[方法]基于酿酒酵母中Srv2序列,通过Blastp比对及关键词搜索,并利用SMART进行保守结构域分析,同时,通过对该菌中AC相关蛋白序列进行理化性质、信号肽、跨膜结构域等分析.[结果]明确该菌含有2个与Srv2同源的AC相关蛋白CgCap1、CgCap2,就理化性质、二级结构等特征方面而言,CgCap1、CgCap2与Srv2有着较大的差异.此外,通过对Srv2与CgCap1、CgCap2及其同源序列进行比对分析,发现CgCap1、CgCap2序列分别与其同源序列及Srv2的C端、N端具有较大的相似性,推测上述序列分别为希金斯炭疽菌AC的C端和N端.[结论]该研究为深入开展该菌AC的研究打下坚实的理论基础.

  4. Shiga Toxin Therapeutics: Beyond Neutralization

    Directory of Open Access Journals (Sweden)

    Gregory Hall

    2017-09-01

    Full Text Available Ribotoxic Shiga toxins are the primary cause of hemolytic uremic syndrome (HUS in patients infected with Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC, a pathogen class responsible for epidemic outbreaks of gastrointestinal disease around the globe. HUS is a leading cause of pediatric renal failure in otherwise healthy children, resulting in a mortality rate of 10% and a chronic morbidity rate near 25%. There are currently no available therapeutics to prevent or treat HUS in STEC patients despite decades of work elucidating the mechanisms of Shiga toxicity in sensitive cells. The preclinical development of toxin-targeted HUS therapies has been hindered by the sporadic, geographically dispersed nature of STEC outbreaks with HUS cases and the limited financial incentive for the commercial development of therapies for an acute disease with an inconsistent patient population. The following review considers potential therapeutic targeting of the downstream cellular impacts of Shiga toxicity, which include the unfolded protein response (UPR and the ribotoxic stress response (RSR. Outcomes of the UPR and RSR are relevant to other diseases with large global incidence and prevalence rates, thus reducing barriers to the development of commercial drugs that could improve STEC and HUS patient outcomes.

  5. Shiga Toxin Therapeutics: Beyond Neutralization

    Science.gov (United States)

    Hall, Gregory; Kurosawa, Shinichiro; Stearns-Kurosawa, Deborah J.

    2017-01-01

    Ribotoxic Shiga toxins are the primary cause of hemolytic uremic syndrome (HUS) in patients infected with Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC), a pathogen class responsible for epidemic outbreaks of gastrointestinal disease around the globe. HUS is a leading cause of pediatric renal failure in otherwise healthy children, resulting in a mortality rate of 10% and a chronic morbidity rate near 25%. There are currently no available therapeutics to prevent or treat HUS in STEC patients despite decades of work elucidating the mechanisms of Shiga toxicity in sensitive cells. The preclinical development of toxin-targeted HUS therapies has been hindered by the sporadic, geographically dispersed nature of STEC outbreaks with HUS cases and the limited financial incentive for the commercial development of therapies for an acute disease with an inconsistent patient population. The following review considers potential therapeutic targeting of the downstream cellular impacts of Shiga toxicity, which include the unfolded protein response (UPR) and the ribotoxic stress response (RSR). Outcomes of the UPR and RSR are relevant to other diseases with large global incidence and prevalence rates, thus reducing barriers to the development of commercial drugs that could improve STEC and HUS patient outcomes. PMID:28925976

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

    Directory of Open Access Journals (Sweden)

    Rachid A. El-Aouar Filho

    2017-05-01

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

  7. 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.

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

    Science.gov (United States)

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-07

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

  9. Perfringolysin O: the underrated Clostridium perfringens toxin?

    OpenAIRE

    Stefanie Verherstraeten; Evy Goossens; Bonnie Valgaeren; Bart Pardon; Leen Timbermont; Freddy Haesebrouck; Richard Ducatelle; Piet Deprez; Kristin R. Wade; Rodney Tweten; Filip Van Immerseel

    2015-01-01

    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 theta 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 membr...

  10. 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.

  11. Botulinum Toxin; Bioterror and Biomedicinal Agent

    Directory of Open Access Journals (Sweden)

    Jiri Patocka

    2006-04-01

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

  12. Natural toxins and their therapeutic potential.

    Science.gov (United States)

    Kapoor, V K

    2010-03-01

    Plants have been extensively investigated for exploring their therapeutic potentials, but there are comparatively scanty reports on drugs derived from animal kingdom, except for hormones. During last decade, the toxins that are used for defense by the animals, have been isolated and found useful tools for physiological and pharmacological studies, besides giving valuable leads to drug development. Toxins with interesting results have been isolated from the venoms of snakes, scorpions, spiders, snails, lizards, frogs and fish. The present review describe about some toxins as drugs and their biological activities. Some fungal, bacterial and marine toxins have also been covered in this article.

  13. 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.

  14. Hemolytic anemia caused by chemicals and toxins

    Science.gov (United States)

    ... This list is not all-inclusive. Alternative Names Anemia - hemolytic - caused by chemicals or toxins References Michel M. Autoimmune and intravascular hemolytic anemias. In: Goldman L, Schafer ...

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

    OpenAIRE

    Natalia López-del Hoyo; Lucrezia Fazioli; Santiago López-Begines; Laura Fernández-Sánchez; Nicolás Cuenca; Jordi Llorens; Pedro de la Villa; Ana Méndez

    2012-01-01

    Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based o...

  16. 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.

  17. Characterization of the acyl-adenylate linked metabolite of mefenamic Acid.

    Science.gov (United States)

    Horng, Howard; Benet, Leslie Z

    2013-03-18

    Mefenamic acid, (MFA), a carboxylic acid-containing nonsteroidal anti-inflammatory drug (NSAID), is metabolized into the chemically reactive conjugates MFA-1-O-acyl-glucuronide (MFA-1-O-G) and MFA-S-acyl-CoA (MFA-CoA), which are both implicated in the formation of MFA-S-acyl-glutathione (MFA-GSH) conjugates, protein-adduct formation, and thus the potential toxicity of the drug. However, current studies suggest that an additional acyl-linked metabolite may be implicated in the formation of MFA-GSH. In the present study, we investigated the ability of MFA to become bioactivated into the acyl-linked metabolite, mefenamyl-adenylate (MFA-AMP). In vitro incubations in rat hepatocytes with MFA (100 μM), followed by LC-MS/MS analyses of extracts, led to the detection of MFA-AMP. In these incubations, the initial rate of MFA-AMP formation was rapid, leveling off at a maximum concentration of 90.1 nM (20 s), while MFA-GSH formation increased linearly, reaching a concentration of 1.7 μM after 60 min of incubation. In comparison, MFA-CoA was undetectable in incubation extracts until the 4 min time point, achieving a concentration of 45.6 nM at the 60 min time point, and MFA-1-O-G formation was linear, attaining a concentration of 42.2 μM after 60 min of incubation. In vitro incubation in buffer with the model nucleophile glutathione (GSH) under physiological conditions showed MFA-AMP to be reactive toward GSH, but 11-fold less reactive than MFA-CoA, while MFA-1-O-G exhibited little reactivity. However, in the presence of glutathione-S-transferase (GST), MFA-AMP mediated formation of MFA-GSH increased 6-fold, while MFA-CoA mediated formation of MFA-GSH only increased 1.4-fold. Collectively, in addition to the MFA-1-O-G, these results demonstrate that mefenamic acid does become bioactivated by acyl-CoA synthetase enzyme(s) in vitro in rat hepatocytes into the reactive transacylating derivatives MFA-AMP and MFA-CoA, both of which contribute to the transacylation of GSH and may

  18. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    Science.gov (United States)

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  19. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    Science.gov (United States)

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-04

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

  20. The metabolic/pH sensor soluble adenylyl cyclase is a tumor suppressor protein

    Science.gov (United States)

    Ramos-Espiritu, Lavoisier; Diaz, Ana; Nardin, Charlee; Saviola, Anthony J.; Shaw, Fiona; Plitt, Tamar; Yang, Xia; Wolchok, Jedd; Pirog, Edyta C.; Desman, Garrett; Sboner, Andrea; Zhang, Tuo; Xiang, Jenny; Merghoub, Taha; Levin, Lonny R.; Buck, Jochen; Zippin, Jonathan H.

    2016-01-01

    cAMP signaling pathways can both stimulate and inhibit the development of cancer; however, the sources of cAMP important for tumorigenesis remain poorly understood. Soluble adenylyl cyclase (sAC) is a non-canonical, evolutionarily conserved, nutrient- and pH-sensing source of cAMP. sAC has been implicated in the metastatic potential of certain cancers, and it is differentially localized in human cancers as compared to benign tissues. We now show that sAC expression is reduced in many human cancers. Loss of sAC increases cellular transformation in vitro and malignant progression in vivo. These data identify the metabolic/pH sensor soluble adenylyl cyclase as a previously unappreciated tumor suppressor protein. PMID:27323809

  1. 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.

  2. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    Science.gov (United States)

    Zippin, Jonathan H.; Farrell, Jeanne; Huron, David; Kamenetsky, Margarita; Hess, Kenneth C.; Fischman, Donald A.; Levin, Lonny R.; Buck, Jochen

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation. PMID:14769862

  3. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    OpenAIRE

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

  4. Interaction Of GCAP1 With Retinal Guanylyl Cyclase And Calcium: Sensitivity to Fatty Acylation

    Directory of Open Access Journals (Sweden)

    Igor V. Peshenko

    2012-02-01

    Full Text Available Guanylyl cyclase activating proteins (GCAP1 are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the non-acylated mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand that is proximal to the myristoyl residue in the three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.

  5. Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins.

    Science.gov (United States)

    Stivala, Craig E; Benoit, Evelyne; Aráoz, Rómulo; Servent, Denis; Novikov, Alexei; Molgó, Jordi; Zakarian, Armen

    2015-03-01

    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 accumulation in shellfish, and chemical stability are important considerations in assessing risk factors for human health. This review article aims to provide an account of chemistry, biology, and toxicology of CI toxins from their discovery to the present day.

  6. 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

    of 0.5 mM CuSO4 to the purified toxin resulted in more intense skin alterations comparable to lesions caused by precipitated culture supernatant diluted 1:10. These results indicated that the activity of the exfoliative toxin was dependent on the presence of Cu2+. Polyclonal and monoclonal antibodies...... were prepared against the exfoliative toxin from strain 1289D-88. The in vivo activity of the exfoliative toxin could be neutralized by antibodies. It was shown that polyclonal as well as monoclonal antibodies only reacted with the toxin produced by two of nine well-defined virulent strains of S...

  7. 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

  8. Tremorgenic Toxin from Penicillium verruculosum

    Science.gov (United States)

    Cole, R. J.; Kirksey, J. W.; Moore, J. H.; Blankenship, B. R.; Diener, U. L.; Davis, N. D.

    1972-01-01

    A new mycotoxin that produces severe tremors and acute toxicity when administered orally or intraperitoneally (ip) to mice and 1-day-old cockerels was obtained from a strain of Penicillium verruculosum Peyronel isolated from peanuts. The ip 50% lethal dose (LD50) of this tremorgen was 2.4 mg/kg in mice and 15.2 mg/kg in chickens. Orally administered LD50 values for the toxin were 126.7 mg/kg in mice and 365.5 mg/kg in chickens. The trivial name „verruculogen” is proposed for this tremorgenic mycotoxin. Physical and chemical characteristics of the mycotoxin are described. PMID:4341967

  9. Tremorgenic toxin from Penicillium veruculosum.

    Science.gov (United States)

    Cole, R J; Kirksey, J W; Moore, J H; Blankenship, B R; Diener, U L; Davis, N D

    1972-08-01

    A new mycotoxin that produces severe tremors and acute toxicity when administered orally or intraperitoneally (ip) to mice and 1-day-old cockerels was obtained from a strain of Penicillium verruculosum Peyronel isolated from peanuts. The ip 50% lethal dose (LD(50)) of this tremorgen was 2.4 mg/kg in mice and 15.2 mg/kg in chickens. Orally administered LD(50) values for the toxin were 126.7 mg/kg in mice and 365.5 mg/kg in chickens. The trivial name "verruculogen" is proposed for this tremorgenic mycotoxin. Physical and chemical characteristics of the mycotoxin are described.

  10. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.

    Science.gov (United States)

    Hubberstey, Andrew V; Mottillo, Emilio P

    2002-04-01

    Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.

  11. 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.

  12. 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.

  13. Genetic reduction of chronic muscle pain in mice lacking calcium/calmodulin-stimulated adenylyl cyclases

    Directory of Open Access Journals (Sweden)

    Petrovic Michele J

    2006-02-01

    Full Text Available Abstract Background The Ca2+/calmodulin-stimulated adenylyl cyclase (AC isoforms AC1 and AC8, couple NMDA receptor activation to cAMP signaling pathways in neurons and are important for development, learning and memory, drug addiction and persistent pain. AC1 and AC8 in the anterior cingulate cortex (ACC and the spinal cord were previously shown to be important in subcutaneous inflammatory pain. Muscle pain is different from cutaneous pain in its characteristics as well as conducting fibers. Therefore, we conducted the present work to test the role of AC1 and AC8 in both acute persistent and chronic muscle pain. Results Using an acute persistent inflammatory muscle pain model, we found that the behavioral nociceptive responses of both the late phase of acute muscle pain and the chronic muscle inflammatory pain were significantly reduced in AC1 knockout (KO and AC1&8 double knockout (DKO mice. Activation of other adenylyl cyclases in these KO mice by microinjection of forskolin into the ACC or spinal cord, but not into the peripheral tissue, rescued the behavioral nociceptive responses. Additionally, intra-peritoneal injection of an AC1 inhibitor significantly reduced behavioral responses in both acute persistent and chronic muscle pain. Conclusion The results of the present study demonstrate that neuronal Ca2+/calmodulin-stimulated adenylyl cyclases in the ACC and spinal cord are important for both late acute persistent and chronic inflammatory muscle pain.

  14. Soluble guanylate cyclase α1-deficient mice: a novel murine model for primary open angle glaucoma.

    Directory of Open Access Journals (Sweden)

    Emmanuel S Buys

    Full Text Available Primary open angle glaucoma (POAG is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1-deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and β1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

  15. Microarray evidence of glutaminyl cyclase gene expression in melanoma: implications for tumor antigen specific immunotherapy

    Directory of Open Access Journals (Sweden)

    Gillis John

    2006-07-01

    Full Text Available Abstract Background In recent years encouraging progress has been made in developing vaccine treatments for cancer, particularly with melanoma. However, the overall rate of clinically significant results has remained low. The present research used microarray datasets from previous investigations to examine gene expression patterns in cancer cell lines with the goal of better understanding the tumor microenvironment. Methods Principal Components Analyses with Promax rotational transformations were carried out with 90 cancer cell lines from 3 microarray datasets, which had been made available on the internet as supplementary information from prior publications. Results In each of the analyses a well defined melanoma component was identified that contained a gene coding for the enzyme, glutaminyl cyclase, which was as highly expressed as genes from a variety of well established biomarkers for melanoma, such as MAGE-3 and MART-1, which have frequently been used in clinical trials of melanoma vaccines. Conclusion Since glutaminyl cyclase converts glutamine and glutamic acid into a pyroglutamic form, it may interfere with the tumor destructive process of vaccines using peptides having glutamine or glutamic acid at their N-terminals. Finding ways of inhibiting the activity of glutaminyl cyclase in the tumor microenvironment may help to increase the effectiveness of some melanoma vaccines.

  16. 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.

  17. 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.

  18. Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

    Science.gov (United States)

    Hatta, S; Amemiya, N; Takemura, H; Ohshika, H

    1995-06-01

    Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

  19. Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of pseudogenes.

    Directory of Open Access Journals (Sweden)

    Jike Cui

    2010-08-01

    Full Text Available Trichomonas vaginalis has an unusually large genome (approximately 160 Mb encoding approximately 60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of approximately 123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs.The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (approximately 46% are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2. Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.

  20. Botulinum Toxin Injections: A Treatment for Muscle Spasms

    Science.gov (United States)

    ... Your Health Resources Drugs, Procedures & Devices Procedures & Devices Botulinum Toxin Injections: A Treatment for Muscle Spasms Botulinum Toxin Injections: A Treatment for Muscle Spasms Drugs, Procedures & ...

  1. Recent insights into Pasteurella multocida toxin and other G-protein-modulating bacterial toxins.

    Science.gov (United States)

    Wilson, Brenda A; Ho, Mengfei

    2010-08-01

    Over the past few decades, our understanding of the bacterial protein toxins that modulate G proteins has advanced tremendously through extensive biochemical and structural analyses. This article provides an updated survey of the various toxins that target G proteins, ending with a focus on recent mechanistic insights in our understanding of the deamidating toxin family. The dermonecrotic toxin from Pasteurella multocida (PMT) was recently added to the list of toxins that disrupt G-protein signal transduction through selective deamidation of their targets. The C3 deamidase domain of PMT has no sequence similarity to the deamidase domains of the dermonecrotic toxins from Escherichia coli (cytotoxic necrotizing factor [CNF]1-3), Yersinia (CNFY) and Bordetella (dermonecrotic toxin). The structure of PMT-C3 belongs to a family of transglutaminase-like proteins, with active site Cys-His-Asp catalytic triads distinct from E. coli CNF1.

  2. [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.

  3. 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.

  4. Tetra- versus Pentavalent Inhibitors of Cholera Toxin

    NARCIS (Netherlands)

    Fu, Ou; Pukin, Aliaksei V.; Quarles Van Ufford, Linda; Branson, Thomas R.; Thies-Weesie, Dominique M E; Turnbull, W. Bruce; Visser, Gerben M.; Pieters, Roland J.

    2015-01-01

    The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface.

  5. 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.

  6. Toxin-Antitoxin Battle in Bacteria

    DEFF Research Database (Denmark)

    Cataudella, Ilaria

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

  7. Tetra- versus Pentavalent Inhibitors of Cholera Toxin

    NARCIS (Netherlands)

    Fu, Ou; Pukin, Aliaksei V.; Quarles Van Ufford, Linda; Branson, Thomas R.; Thies-Weesie, Dominique M E; Turnbull, W. Bruce; Visser, Gerben M.; Pieters, Roland J.

    2015-01-01

    The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface.

  8. The Ins and Outs of Anthrax Toxin

    Science.gov (United States)

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

    2016-01-01

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

  9. Botulinum toxin — therapeutic effect in cosmetology

    Directory of Open Access Journals (Sweden)

    Morrison A.V.

    2016-09-01

    Full Text Available This review presents the data from published literatures and the research works conducted by the authors about mechanisms of action of botulinum toxin and its use in the practical medicine (particularly in dermatology and cosmetology. Indications and contraindications of botulinum toxin use in cosmetology are also considered in this work.

  10. 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...

  11. Bacterial toxins and the immune system

    Science.gov (United States)

    Galán, Jorge E.

    2005-01-01

    Microorganisms that cause persistent infection often exhibit specific adaptations that allow them to avoid the adaptive immune response. Recently, several bacterial toxins have been shown in vitro to disrupt immune cell functions. However, it remains to be established whether these activities are relevant during infection and whether these toxins have specifically evolved to disrupt the adaptive immune system. PMID:15699067

  12. Revealing the Achilles heel of bacterial toxins.

    Science.gov (United States)

    Schröder, Jens-Michael

    2014-11-20

    In addition to having antimicrobial properties, defensins inactivate various structurally unrelated bacterial toxins by a yet unknown manner. In this issue of Immunity, Kudryashova et al. (2014b) provide insights into mechanisms by which human ?-defensins destabilize and inactivate bacterial toxins. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. DETECTION OF BACTERIAL TOXINS WITH MONOSACCHARIDE ARRAYS

    Science.gov (United States)

    Ngundi, Miriam M.; Taitt, Chris R.; McMurry, Scott A.; Kahne, Daniel; Ligler, Frances S.

    2006-01-01

    A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach to and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml. PMID:15946840

  14. 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.

  15. Toxin-Antitoxin Battle in Bacteria

    DEFF Research Database (Denmark)

    Cataudella, Ilaria

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

  16. Target-Driven Evolution of Scorpion Toxins.

    Science.gov (United States)

    Zhang, Shangfei; Gao, Bin; Zhu, Shunyi

    2015-10-07

    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.

  17. Measuring Immunoglobulin G Antibodies to Tetanus Toxin, Diphtheria Toxin, and Pertussis Toxin with Single-Antigen Enzyme-Linked Immunosorbent Assays and a Bead-Based Multiplex Assay▿

    OpenAIRE

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz

    2008-01-01

    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assa...

  18. Clostridium botulinum Type E Toxins Bind to Caco-2 Cells by a Different Mechanism from That of Type A Toxins

    Directory of Open Access Journals (Sweden)

    Zhang,Kai

    2012-06-01

    Full Text Available Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S and a non-toxic non-hemagglutinin (NTNH. The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA, and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H and light (L chains by a protease(s in some strains, and the H chain has 2 domains, the N-terminus (Hn and C-terminus (Hc. It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin.

  19. 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...

  20. 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.

  1. Stealth and mimicry by deadly bacterial toxins

    DEFF Research Database (Denmark)

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

    2006-01-01

    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......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...... 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....

  2. 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.

  3. Toxins and secretion systems of Photorhabdus luminescens.

    Science.gov (United States)

    Rodou, Athina; Ankrah, Dennis O; Stathopoulos, Christos

    2010-06-01

    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.

  4. Bacterial toxins and Multiple Sclerosis.

    Science.gov (United States)

    Gay, Frederick

    2007-11-15

    The primary pathogenetic mechanism responsible for the distinctive demyelinating lesions in the Central Nervous System (CNS) in Multiple Sclerosis (MS), first described in remarkable detail by Charcot more than 170 years ago, remains one of the most baffling conundrums in medicine. A possible role for bacterial cell molecules and transportable proteins in the pathogenesis of MS is reviewed. The ability of bacterial toxins to distort immunity and to cause distinctive toxic damage in the nervous system is discussed in the light of largely forgotten data linking bacterial nasopharyngeal infections with optic neuritis, optochiasmatic arachnoiditis and MS. While the blood-brain barrier substantially protects the CNS from hematogenous toxins, there is a route by which the barrier may be by-passed. Data is reviewed which shows that the CSF and extra-cellular fluid circulation is bi-directionally linked to the lymphatic drainage channels of the nasopharyngeal mucosa. While this provides a facility by which the CNS may mount immunological responses to antigenic challenges from within, it is also a route by which products of nasopharyngeal infection may drain into the CNS and be processed by the immune cells of the meninges and Virchow-Robin perivascular spaces. If potentially toxic bacterial products are identified in early MS tissues at these sites, this would provide an entirely new insight into the pathogenetic mechanisms of this frustratingly enigmatic disease.

  5. Laboratory Evaluation of Adenylate Energy Charge as a Test for Stress in Mytilus edulis and Nephtys incisa Treated with Dredged Material.

    Science.gov (United States)

    1985-02-01

    concentrations of three adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), which are...that all trace metals but iron were eliminated and the concentration of the vitamins thiamin and B12 were doubled. Adenylate Extraction 13. The adductor

  6. Effect of glucocorticoid on prostaglandin E1 mediated cyclic AMP formation by vascular smooth muscle cells.

    Science.gov (United States)

    Yasunari, K; Kohno, M; Murakawa, K; Yokokawa, K; Takeda, T

    1988-12-01

    The effect of glucocorticoid on the prostaglandin E1 (PGE1)-mediated cyclic AMP (cAMP) formation by vascular smooth muscle cells (VSMC) from renal arteries (RA) was studied in rats. Dexamethasone (DEX) at concentrations ranging from 10(-10) to approximately 10(-8) mol/l dose-dependently potentiates the PGE1-mediated response. This facilitation began at 6 h and reached its maximum after 24 h of DEX administration. Aldosterone (10(-6) mol/l) did not affect the dose-response curve of PGE1. Inhibitors of protein and RNA synthesis blocked this glucocorticoid effect. The basal activity of adenylate cyclase in DEX-treated cells was twice as high as in control cells. Treatment of VSMC with DEX increased cholera toxin- and pertussis toxin-stimulated adenylate cyclase activity. DEX treatment also augments forskolin-stimulated adenylate cyclase activity. These results suggest that DEX increases PGE1-mediated cAMP formation of VSMC from RA through a mechanism that involves the induction of protein synthesis, and that the activation of the catalytic unit may play some role in this facilitating process.

  7. Evaluation of antidiphtheria toxin nanobodies

    Directory of Open Access Journals (Sweden)

    Ghada H Shaker

    2010-06-01

    Full Text Available Ghada H ShakerDepartment of Microbiology and Immunology, Faculty of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi ArabiaAbstract: Nanobodies are the smallest fragments of naturally occurring single-domain antibodies that have evolved to be fully functional in the absence of a light chain. Conventional antibodies are glycoproteins comprising two heavy and two light chains. Surprisingly, all members of the Camelidae family possess a fraction of antibodies devoid of both light chains and the first constant domain. These types of antibodies are known as heavy-chain antibody (HcAb nanobodies. There are three subclasses of IgG in dromedaries, namely IgG1, IgG2, and IgG3 of which IgG2 and IgG3 are of the HcAb type. These heavy chain antibodies constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. In the present work, the different IgG subclasses from an immunized camel (Camelus dromedarius with divalent diphtheria-tetanus vaccine were purified using their different affinity for protein A and protein G and their absorbance measured at 280 nm. Purity control and characterization by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis of IgG subclasses was done under reducing conditions. Protein bands were visualized after staining with Coomassie Blue, showing two bands at 50 kDa and 30 kDa for IgG1, while IgG2 and IgG3 produced only one band at 46 kDa and 43 kDa, respectively. An enzyme-linked immunosorbent assay test using diphtheria toxin and purified IgG subclasses from the immunized camel were performed to evaluate their efficiency. Compared with conventional IgG1, heavy chain antibodies (nanobodies were shown to be more efficient in binding to diphtheria toxin antigen. This study revealed the possibility of using IgG2 and IgG3 nanobodies as an effective antitoxin for the treatment of diphtheria in humans.Keywords: camel, heavy chain antibody, HcAb, nanobodies, immunoglobulin, Ig

  8. Single toxin dose-response models revisited

    Science.gov (United States)

    Glaholt, SP; Kyker-Snowman, E; Shaw, JR; Chen, CY

    2016-01-01

    The goal of this paper is to offer a rigorous analysis of the sigmoid shape single toxin dose-response relationship. The toxin efficacy function is introduced and four special points, including maximum toxin efficacy and inflection points, on the dose-response curve are defined. The special points define three phases of the toxin effect on mortality: (1) toxin concentrations smaller than the first inflection point or (2) larger then the second inflection point imply low mortality rate, and (3) concentrations between the first and the second inflection points imply high mortality rate. Probabilistic interpretation and mathematical analysis for each of four models, Hill, logit, probit, and Weibull is provided. Two general model extensions are introduced: (1) the multi-target hit model that accounts for the existence of several vital receptors affected by the toxin, and (2) model with a nonzero mortality at zero concentration to account for natural mortality. Special attention is given to statistical estimation in the framework of the generalized linear model with the binomial dependent variable as the mortality count in each experiment, contrary to the widespread nonlinear regression treating the mortality rate as continuous variable. The models are illustrated using standard EPA Daphnia acute (48 hours) toxicity tests with mortality as a function of NiCl or CuSO4 toxin. PMID:27847315

  9. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    Science.gov (United States)

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan

    2009-10-01

    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  10. The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

    Science.gov (United States)

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-02-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

  11. 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.

  12. Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

    Science.gov (United States)

    Quinn, Sierra N; Graves, Sarai H; Dains-McGahee, Clayton; Friedman, Emilee M; Hassan, Humma; Witkowski, Piotr; Sabbatini, Maria E

    2017-04-01

    Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc.

  13. Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation.

    Science.gov (United States)

    Shima, F; Okada, T; Kido, M; Sen, H; Tanaka, Y; Tamada, M; Hu, C D; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    2000-01-01

    Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.

  14. Continuous Production of Clostridium tetani Toxin

    Science.gov (United States)

    Zacharias, Bengt; Björklund, Marianne

    1968-01-01

    The continuous production of Clostridium tetani toxin has been carried out in a 1-liter stirred culture vessel for as long as 65 days. Toxin production of approximately 120 flocculating units per ml was maintained with a dilution rate of 0.125 hr-1, a temperature of 34 C, a pH of 7.4, and the addition to the medium of 0.1 g of potassium chloride per liter. The average minimal lethal intraperitoneal dose of the toxin in mice was approximately 106 per ml. PMID:4865906

  15. Clostridium perfringens type A–E toxin plasmids

    Science.gov (United States)

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  16. Engineering modified Bt toxins to counter insect resistance.

    Science.gov (United States)

    Soberón, Mario; Pardo-López, Liliana; López, Idalia; Gómez, Isabel; Tabashnik, Bruce E; Bravo, Alejandra

    2007-12-07

    The evolution of insect resistance threatens the effectiveness of Bacillus thuringiensis (Bt) toxins that are widely used in sprays and transgenic crops. Resistance to Bt toxins in some insects is linked with mutations that disrupt a toxin-binding cadherin protein. We show that susceptibility to the Bt toxin Cry1Ab was reduced by cadherin gene silencing with RNA interference in Manduca sexta, confirming cadherin's role in Bt toxicity. Native Cry1A toxins required cadherin to form oligomers, but modified Cry1A toxins lacking one alpha-helix did not. The modified toxins killed cadherin-silenced M. sexta and Bt-resistant Pectinophora gossypiella that had cadherin deletion mutations. Our findings suggest that cadherin promotes Bt toxicity by facilitating toxin oligomerization and demonstrate that the modified Bt toxins may be useful against pests resistant to standard Bt toxins.

  17. 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.

  18. Fold modulating function: bacterial toxins to functional amyloids

    OpenAIRE

    Adnan Khawaja Syed; Blaise R. Boles

    2014-01-01

    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...

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

    Science.gov (United States)

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

    2016-09-01

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

  20. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    Energy Technology Data Exchange (ETDEWEB)

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S. (Biosciences Division); (Univ. of Nevada); (Alliance Protein Labs.)

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.