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Sample records for amp

  1. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  2. AMP language reference manual

    International Nuclear Information System (INIS)

    The use of a program for symbolic calculations named AMP (Algebraic Manipulation Program) is described. Its main features are: high speed heart for usual algebraic calculations; conversational capability; derivation, substitutions, matricial calculus, expansions, non commutative algebrae...; possibility to define new symbols and associated rules; possibility to create and use external libraries; written for IBM/370 like computers

  3. Sleutelmag en amp

    Directory of Open Access Journals (Sweden)

    P. J. Rossouw

    1985-06-01

    Full Text Available As die Heidelbergse Kategismus (Sondag 31 handel oor die sleutels van die Koninkryk, dan word die betrokkenheid van die ampte slegs per implikasie veronderstel. Meer eksplisiet word die amp en sleutelmag in die Nederlandse Geloofsbelydenis gekoppel; “ons glo dat hierdie ware kerk ooreenkomstig die geestelike bestuurswyse wat ons Here ons in sy Woord geleer het, geregeer moet word... hulle (moet sorg dra dat die ware godsdiens onderhou (word, die ware leer orals versprei, die oortreders op geestelike wyse vermaan en in toom gehou (word... (N.G.B. art. 30 en: “Ons glo verder dat die regeerders van die kerk, al is dit nuttig en goed om onder mekaar ’n bepaalde orde tot instandhouding van die liggaam van die kerk in te stel en te handhaaf, tog noukeurig moet oppas om nie af te wyk van wat Christus, ons enigste Meester, vir ons ingestel het nie... Ons aanvaar derhalwe slegs wat kan dien om eendrag en eenheid te bewaar en te bevorder, en om alles in gehoorsaamheid van God te onderhou. Daarvoor is nodig die ban en alles wat daarmee saamhang, toegepas ooreenkomstig die Woord van God” (N.G.B. art. 32.

  4. Assisted Medical Procedures (AMP) Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Documentation and Development: The AMP was initially being developed as part the Advanced Integrated Clinical System (AICS)-Guided Medical Procedure System for the...

  5. Agile manufacturing prototyping system (AMPS)

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, P.

    1998-05-09

    The Agile Manufacturing Prototyping System (AMPS) is being integrated at Sandia National Laboratories. AMPS consists of state of the industry flexible manufacturing hardware and software enhanced with Sandia advancements in sensor and model based control; automated programming, assembly and task planning; flexible fixturing; and automated reconfiguration technology. AMPS is focused on the agile production of complex electromechanical parts. It currently includes 7 robots (4 Adept One, 2 Adept 505, 1 Staubli RX90), conveyance equipment, and a collection of process equipment to form a flexible production line capable of assembling a wide range of electromechanical products. This system became operational in September 1995. Additional smart manufacturing processes will be integrated in the future. An automated spray cleaning workcell capable of handling alcohol and similar solvents was added in 1996 as well as parts cleaning and encapsulation equipment, automated deburring, and automated vision inspection stations. Plans for 1997 and out years include adding manufacturing processes for the rapid prototyping of electronic components such as soldering, paste dispensing and pick-and-place hardware.

  6. Pseudomonas aeruginosa β-lactamase induction requires two permeases, AmpG and AmpP

    Directory of Open Access Journals (Sweden)

    Schneper Lisa

    2010-12-01

    Full Text Available Abstract Background In Enterobacteriaceae, β-lactam antibiotic resistance involves murein recycling intermediates. Murein recycling is a complex process with discrete steps taking place in the periplasm and the cytoplasm. The AmpG permease is critical to this process as it transports N-acetylglucosamine anhydrous N-acetylmuramyl peptides across the inner membrane. In Pseudomonadaceae, this intrinsic mechanism remains to be elucidated. Since the mechanism involves two cellular compartments, the characterization of transporters is crucial to establish the link. Results Pseudomonas aeruginosa PAO1 has two ampG paralogs, PA4218 (ampP and PA4393 (ampG. Topology analysis using β-galactosidase and alkaline phosphatase fusions indicates ampP and ampG encode proteins which possess 10 and 14 transmembrane helices, respectively, that could potentially transport substrates. Both ampP and ampG are required for maximum expression of β-lactamase, but complementation and kinetic experiments suggest they act independently to play different roles. Mutation of ampG affects resistance to a subset of β-lactam antibiotics. Low-levels of β-lactamase induction occur independently of either ampP or ampG. Both ampG and ampP are the second members of two independent two-gene operons. Analysis of the ampG and ampP operon expression using β-galactosidase transcriptional fusions showed that in PAO1, ampG operon expression is β-lactam and ampR-independent, while ampP operon expression is β-lactam and ampR-dependent. β-lactam-dependent expression of the ampP operon and independent expression of the ampG operon is also dependent upon ampP. Conclusions In P. aeruginosa, β-lactamase induction occurs in at least three ways, induction at low β-lactam concentrations by an as yet uncharacterized pathway, at intermediate concentrations by an ampP and ampG dependent pathway, and at high concentrations where although both ampP and ampG play a role, ampG may be of greater

  7. The AzTEC Mathematics Project (AMP).

    Science.gov (United States)

    Johnson, Gae R.

    The AzTEC Mathematics Project (AMP) is a statewide partnership among Arizona's Regents universities and state community colleges, partner school districts, and economic communities. AzTec is committed to preparing highly qualified K-12 mathematics and science teachers. AMP targeted Native American teachers and teachers of Native American students…

  8. Warmer amps for the LHC

    CERN Multimedia

    Anaïs Schaeffer

    2012-01-01

    CERN is working together with an Italian company to develop superconducting cables that can function at temperatures of up to 25 K (-248°C). This will make it possible to move LHC magnet power supplies out of the tunnel, protecting them from exposure to the showers of very high-energy particles produced by the accelerator.   Figure 1: devices of this type, which measure approximately 10 metres in length, are inserted between the accelerating magnets at different points along the LHC. When it comes to consuming electricity, the magnets that steer particles through large accelerators can be characterised with just one word: greedy. For the LHC, the total current can reach 1.5 million amps. At the present time, this current is brought in via copper cables of up to 10 cm in diameter. In the tunnel, these cables connect the current leads - which provide the transition between the ambient-temperature cables and the magnets in their bath of superfluid helium - to the power supply. In the a...

  9. C++ Coding Standards for the AMP Project

    Energy Technology Data Exchange (ETDEWEB)

    Evans, Thomas M [ORNL; Clarno, Kevin T [ORNL

    2009-09-01

    This document provides an initial starting point to define the C++ coding standards used by the AMP nuclear fuel performance integrated code project and a part of AMP's software development process. This document draws from the experiences, and documentation [1], of the developers of the Marmot Project at Los Alamos National Laboratory. Much of the software in AMP will be written in C++. The power of C++ can be abused easily, resulting in code that is difficult to understand and maintain. This document gives the practices that should be followed on the AMP project for all new code that is written. The intent is not to be onerous but to ensure that the code can be readily understood by the entire code team and serve as a basis for collectively defining a set of coding standards for use in future development efforts. At the end of the AMP development in fiscal year (FY) 2010, all developers will have experience with the benefits, restrictions, and limitations of the standards described and will collectively define a set of standards for future software development. External libraries that AMP uses do not have to meet these requirements, although we encourage external developers to follow these practices. For any code of which AMP takes ownership, the project will decide on any changes on a case-by-case basis. The practices that we are using in the AMP project have been in use in the Denovo project [2] for several years. The practices build on those given in References [3-5]; the practices given in these references should also be followed. Some of the practices given in this document can also be found in [6].

  10. Changes in nephrogenous cyclic AMP excretion and plasma cyclic AMP following treatment of hyperthyroidism.

    Science.gov (United States)

    Naafs, M A; van der Velden, P C; Fischer, H R; Koorevaar, G; van Duin, S; Hackeng, W H; Schopman, W; Silberbusch, J

    1984-08-01

    Plasma cyclic AMP (PcAMP) concentration and the excretion of cyclic AMP/dl GF were estimated in 11 thyrotoxic patients before and after medical treatment. PcAMP concentrations were significantly higher during hyperthyroidism (2.30 +/- 0.69 vs 1.88 +/- 0.71 nmol/dl; P less than 0.05), and total urinary cyclic AMP (TcAMP) excretion showed no significant changes (3.24 +/- 0.64 vs 3.44 +/- 1.77 nmol/dl GF). Nephrogenous (NcAMP) excretion rose significantly (1.00 +/- 0.82 vs 1.68 +/- 1.31 mmol/dl GF; P less than 0.025). The increase in NcAMP excretion correlated significantly with the decrease in serum T3 levels (r = -0.46; P less than 0.05). Serum iPTH levels showed no significant change. Both the serum Ca, corrected for serum total protein and TmPO4/GFR declined after treatment (respectively 2.44 +/- 0.13 vs 2.33 +/- 0.08 mmol/l; P less than 0.05 and 1.18 +/- 0.29 vs 1.05 +/- 0.22 mmol/l; P less than 0.05). It is concluded that the rise in NcAMP excretion corroborates the concept of increasing parathyroid activity following the treatment of hyperthyroidism. PMID:6206676

  11. S-AMP for non-linear observation models

    DEFF Research Database (Denmark)

    Cakmak, Burak; Winther, Ole; Fleury, Bernard H.

    2015-01-01

    Recently we presented the S-AMP approach, an extension of approximate message passing (AMP), to be able to handle general invariant matrix ensembles. In this contribution we extend S-AMP to non-linear observation models. We obtain generalized AMP (GAMP) as the special case when the measurement...

  12. cAMP signalling meets mitochondrial compartments.

    Science.gov (United States)

    Lefkimmiatis, Konstantinos

    2014-04-01

    Mitochondria are highly dynamic organelles comprising at least three distinct areas, the OMM (outer mitochondrial membrane), the IMS (intermembrane space) and the mitochondrial matrix. Physical compartmentalization allows these organelles to host different functional domains and therefore participate in a variety of important cellular actions such as ATP synthesis and programmed cell death. In a surprising homology, it is now widely accepted that the ubiquitous second messenger cAMP uses the same stratagem, compartmentalization, in order to achieve the characteristic functional pleiotropy of its pathway. Accumulating evidence suggests that all the main mitochondrial compartments contain segregated cAMP cascades; however, the regulatory properties and functional significance of such domains are not fully understood and often remain controversial issues. The present mini-review discusses our current knowledge of how the marriage between mitochondrial and cAMP compartmentalization is achieved and its effects on the biology of the cell. PMID:24646228

  13. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller;

    2008-01-01

    Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for c...

  14. Multiple facets of cAMP signalling and physiological impact : cAMP compartmentalization in the lung

    NARCIS (Netherlands)

    Oldenburger, Anouk; Maarsingh, Harm; Schmidt, Martina

    2012-01-01

    Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelli

  15. The Applied Mathematics for Power Systems (AMPS)

    Energy Technology Data Exchange (ETDEWEB)

    Chertkov, Michael [Los Alamos National Laboratory

    2012-07-24

    Increased deployment of new technologies, e.g., renewable generation and electric vehicles, is rapidly transforming electrical power networks by crossing previously distinct spatiotemporal scales and invalidating many traditional approaches for designing, analyzing, and operating power grids. This trend is expected to accelerate over the coming years, bringing the disruptive challenge of complexity, but also opportunities to deliver unprecedented efficiency and reliability. Our Applied Mathematics for Power Systems (AMPS) Center will discover, enable, and solve emerging mathematics challenges arising in power systems and, more generally, in complex engineered networks. We will develop foundational applied mathematics resulting in rigorous algorithms and simulation toolboxes for modern and future engineered networks. The AMPS Center deconstruction/reconstruction approach 'deconstructs' complex networks into sub-problems within non-separable spatiotemporal scales, a missing step in 20th century modeling of engineered networks. These sub-problems are addressed within the appropriate AMPS foundational pillar - complex systems, control theory, and optimization theory - and merged or 'reconstructed' at their boundaries into more general mathematical descriptions of complex engineered networks where important new questions are formulated and attacked. These two steps, iterated multiple times, will bridge the growing chasm between the legacy power grid and its future as a complex engineered network.

  16. The Monge-Ampère equation

    CERN Document Server

    Gutiérrez, Cristian E

    2016-01-01

    Now in its second edition, this monograph explores the Monge-Ampère equation and the latest advances in its study and applications. It provides an essentially self-contained systematic exposition of the theory of weak solutions, including regularity results by L. A. Caffarelli. The geometric aspects of this theory are stressed using techniques from harmonic analysis, such as covering lemmas and set decompositions. An effort is made to present complete proofs of all theorems, and examples and exercises are offered to further illustrate important concepts. Some of the topics considered include generalized solutions, non-divergence equations, cross sections, and convex solutions. New to this edition is a chapter on the linearized Monge-Ampère equation and a chapter on interior Hölder estimates for second derivatives. Bibliographic notes, updated and expanded from the first edition, are included at the end of every chapter for further reading on Monge-Ampère-type equations and their diverse applications in th...

  17. S-AMP: Approximate Message Passing for General Matrix Ensembles

    DEFF Research Database (Denmark)

    Cakmak, Burak; Winther, Ole; Fleury, Bernard H.

    2014-01-01

    We propose a novel iterative estimation algorithm for linear observation models called S-AMP. The fixed points of S-AMP are the stationary points of the exact Gibbs free energy under a set of (first- and second-) moment consistency constraints in the large system limit. S-AMP extends the...... approximate message-passing (AMP) algorithm to general matrix ensembles with a well-defined large system size limit. The generalization is based on the S-transform (in free probability) of the spectrum of the measurement matrix. Furthermore, we show that the optimality of S-AMP follows directly from its...

  18. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia

    Science.gov (United States)

    Perez, Dominique R.; Smagley, Yelena; Garcia, Matthew; Carter, Mark B.; Evangelisti, Annette; Matlawska-Wasowska, Ksenia; Winter, Stuart S.; Sklar, Larry A.; Chigaev, Alexandre

    2016-01-01

    Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3′-5′-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing. PMID:27129155

  19. Phosphodiesterase 4 and compartmentalization of cyclic AMP signaling

    Institute of Scientific and Technical Information of China (English)

    WANG ZhengChao; SHI FangXiong

    2007-01-01

    Cyclic AMP (cAMP), as a second messenger, plays a critical role in cellular signaling transduction. However, it is not clear how this apparently identical cAMP signal induces divergent physiological responses. The potential explanation that cAMP signaling is compartmentalized was proposed by Buxton and Brunton twenty years ago. Compartmentalization of cAMP signaling allows spatially distinct pools of protein kinase A (PKA) to be differently activated. Research on cAMP signaling has regained impetus in many fields of life sciences due to the progress in understanding cAMP signaling complexity and functional diversity. The cAMP/PKA signaling compartments are maintained by A-kinase anchoring proteins (AKAPs) which bind PKA and other signaling proteins, and by PDEs which hydrolyse cAMP and thus terminate PKA activity. PDE4 enzymes belong to PDE superfamily and stand at a crossroad that allows them to integrate various signaling pathways with that of cAMP in spatially distinct compartments. In the current review, the nomenclature, taxonomy and gene expression of PDE4, and the system and region of its effect are described. In addition, the idiographic molecules, mechanisms, and regulation models of PDE4 are summarized. Furthermore, the important roles PDE4 plays in the maturation of rat granulosa cells and cAMP signaling compartmentalization are discussed.

  20. Optimal Design for Reactivity Ratio Estimation: A Comparison of Techniques for AMPS/Acrylamide and AMPS/Acrylic Acid Copolymerizations

    Directory of Open Access Journals (Sweden)

    Alison J. Scott

    2015-11-01

    Full Text Available Water-soluble polymers of acrylamide (AAm and acrylic acid (AAc have significant potential in enhanced oil recovery, as well as in other specialty applications. To improve the shear strength of the polymer, a third comonomer, 2-acrylamido-2-methylpropane sulfonic acid (AMPS, can be added to the pre-polymerization mixture. Copolymerization kinetics of AAm/AAc are well studied, but little is known about the other comonomer pairs (AMPS/AAm and AMPS/AAc. Hence, reactivity ratios for AMPS/AAm and AMPS/AAc copolymerization must be established first. A key aspect in the estimation of reliable reactivity ratios is design of experiments, which minimizes the number of experiments and provides increased information content (resulting in more precise parameter estimates. However, design of experiments is hardly ever used during copolymerization parameter estimation schemes. In the current work, copolymerization experiments for both AMPS/AAm and AMPS/AAc are designed using two optimal techniques (Tidwell-Mortimer and the error-in-variables-model (EVM. From these optimally designed experiments, accurate reactivity ratio estimates are determined for AMPS/AAm (rAMPS = 0.18, rAAm = 0.85 and AMPS/AAc (rAMPS = 0.19, rAAc = 0.86.

  1. Directed evolution of the Escherichia coli cAMP receptor protein at the cAMP pocket.

    Science.gov (United States)

    Gunasekara, Sanjiva M; Hicks, Matt N; Park, Jin; Brooks, Cory L; Serate, Jose; Saunders, Cameron V; Grover, Simranjeet K; Goto, Joy J; Lee, Jin-Won; Youn, Hwan

    2015-10-30

    The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP.

  2. Internal gastargets in AmPS

    Science.gov (United States)

    Kaan, A. P.; Postma, O.; van den Brand, J. F. J.; van Leeuwen, E.; Doets, M.; Kraan, M.

    1997-05-01

    Internal gas targets in AmPS A.P. Kaan, O. Postma, J.F.J. van den Brand, E. van Leeuwen, M. Doets, M. Kra= an National Institute for Nuclear Physics and High Energy Physics; Kruislaan 409; 1098 SJ Amsterdam; Holland In the Amsterdam Puls Stretcher/storage ring AmPS(1 GeV electrons), we designed a set-up in order to accommodate a gas target with a density of 1016 mol/cm2. The storage cell needed for this purpose is a aluminium tube with a length of 40 cm, a diameter of 15 mm and a wall thickness of 25 =B5m. Three sets of conductance limiters on both sides of the target, combined with dry turbopumps are designed to be used as differential pumping stations. These limiters cause discontinuities in the beam path and must therefor be retractable and radio frequency compatible in both positions. Low =B5 materials must be used because of the depolarisation effects of changing magnetic fields. The calculations show that the flow resistance's are sufficient to reduce the load of the getter pumps to a level with which the lifetime of the pump elements remain acceptable. The design of the mechanics and the vacuum system will be explained. Recent results from the measurements after installation in combination with the influence on the lifetime on the beam will be presented

  3. Cardiac cAMP: production, hydrolysis, modulation and detection

    OpenAIRE

    Cédric eBOULARAN; Céline eGALES

    2015-01-01

    Cyclic adenosine 3’,5’-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors’ signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefl...

  4. Cardiac cAMP: production, hydrolysis, modulation and detection

    OpenAIRE

    Boularan, Cédric; Gales, Céline

    2015-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefl...

  5. Cardiac cAMP: production, hydrolysis, modulation and detection.

    Science.gov (United States)

    Boularan, Cédric; Gales, Céline

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability. PMID:26483685

  6. Antagonists of chemoattractants reveal separate receptors for cAMP, folic acid and pterin in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Wit, René J.W. de; Konijn, Theo M.

    1982-01-01

    Adenosine 3’,5’-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3’-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3’-amino-cAMP on the ch

  7. Control of bacterial exoelectrogenesis by c-AMP-GMP.

    Science.gov (United States)

    Nelson, James W; Sudarsan, Narasimhan; Phillips, Grace E; Stav, Shira; Lünse, Christina E; McCown, Phillip J; Breaker, Ronald R

    2015-04-28

    Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

  8. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    DEFF Research Database (Denmark)

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.;

    2015-01-01

    Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors...... as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult...... zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance...

  9. The Popeye Domain Containing Genes and cAMP Signaling

    Directory of Open Access Journals (Sweden)

    Thomas Brand

    2014-05-01

    Full Text Available 3'-5'-cyclic adenosine monophosphate (cAMP is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs. Initially, it was thought that protein kinase A (PKA exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC and hyperpolarizing cyclic nucleotide-gated (HCN channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.

  10. Imaging alterations of cardiomyocyte cAMP microdomains in disease

    Directory of Open Access Journals (Sweden)

    Alexander eFroese

    2015-08-01

    Full Text Available 3’,5’-cyclic adenosine monophosphate (cAMP is an important second messenger which regulates heart function by acting in distinct subcellular microdomains. Recent years have provided deeper mechanistic insights into compartmentalized cAMP signaling and its link to cardiac disease. In this mini review, we summarize newest developments in this field achieved by cutting-edge biochemical and biophysical techniques. We further compile the data from different studies into a bigger picture of so far uncovered alterations in cardiomyocyte cAMP microdomains which occur in compensated cardiac hypertrophy and chronic heart failure. Finally, future research directions and translational perspectives are briefly discussed.

  11. Multiple Facets of cAMP Signalling and Physiological Impact: cAMP Compartmentalization in the Lung

    Directory of Open Access Journals (Sweden)

    Martina Schmidt

    2012-11-01

    Full Text Available Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD. Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myofibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP superfamily and IQ motif containing GTPase activating protein (IQGAPs. In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target.

  12. Isolation of an inactive bovine heart cAMP-dependent protein kinase holoenzyme containing bound cAMP

    International Nuclear Information System (INIS)

    Bovine heart Type II cAMP-dependent protein kinase (PK) was purified to homogeneity as determined by SDS-PAGE. The purification steps were DEAE-cellulose, ammonium sulfate precipitation, phenyl-Sepharose, alumina C-γ, and HPLC-DEAE. The last step resolved two distinct peaks of cAMP dependent kinase activity (activity ratios = 0.05 - 0.10) eluting at approximately 250 (Peak 1) and 275 (Peak 2) mM NaCl. When subjected to HPLC-gel permeation they had the same Stoke's radii. Linear sucrose gradients gave S/sub 20,w/ values of 7.73 S and 7.25 S for Peaks 1 and 2, respectively. It was determined by integrating the areas of scanned SDS-PAGE bands that regulatory and catalytic subunits were present in equimolar amounts in both Peaks 1 and 2. The ratios of equilibrium [3H] cAMP binding to kinase activity for the two peaks were the same. cAMP was present in trace amounts in Peak 1 but in stoichiometric amounts in Peak 2 (between 25 and 75 % saturation of cAMP binding sites). Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP, 3H] cAMP binding and in kinase activation (as indicated by a larger Hill coefficient) of Peak 2 relative to Peak 1 also supported the presence of cAMP in Peak 2. In conclusion, two forms of inactive PK have been isolated, one of which is a ternary complex of PK holoenzyme and cAMP. This complex could represent a cellular form of the enzyme which is primed for activation

  13. Conditions that alter intracellular cAMP levels affect expression of the cAMP phosphodiesterase gene in Dictyostelium.

    OpenAIRE

    Riley, B B; Barclay, S L

    1990-01-01

    We examined expression of the Dictyostelium cAMP phosphodiesterase (PDE) gene under conditions that alter intracellular cAMP levels during in vitro differentiation of wild-type strain V12M2 and a sporogenous derivative, HB200. In control cultures, cellular PDE activity peaked at 6 hr and declined by 8 hr, while secreted PDE activity continued to increase through 8 hr. Lowering intracellular cAMP levels with caffeine or progesterone increased cellular and secreted PDE activities 2-fold, increa...

  14. 21 CFR 862.1230 - Cyclic AMP test system.

    Science.gov (United States)

    2010-04-01

    .... Cyclic AMP measurements are used in the diagnosis and treatment of endocrine disorders, including... and treatment of Graves' disease (a disorder of the thyroid) and in the differentiation of causes...

  15. Cardiac cAMP: production, hydrolysis, modulation and detection

    Directory of Open Access Journals (Sweden)

    Cédric eBOULARAN

    2015-10-01

    Full Text Available Cyclic adenosine 3’,5’-monophosphate (cAMP modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors’ signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability.

  16. Amped Up! - Volume 1, No. 3, May/June 2015

    Energy Technology Data Exchange (ETDEWEB)

    None

    2015-05-01

    Welcome to the latest issue of our bimonthly newsletter, Amped Up!, highlighting the initiatives, events and technologies in the Office of Energy Efficiency and Renewable Energy that influence change.

  17. ^amp;+^amp;-p Electroproduction Cross Sections off Protons in the Second Resonance Region

    Science.gov (United States)

    Fedotov, Gleb; Gothe, Ralf; Mokeev, Victor

    2013-04-01

    In this talk we present preliminary ^amp;+^amp;-p electroproduction cross sections off protons in the kinematical area of W from 1.4 to 1.8 GeV and Q^2 from 0.4 to 1.1 GeV^2. Our kinematical coverage in part overlap with previous CLAS measurements, but offers more than a factor six finer binning in Q^2. The physics analysis of these data within the framework of the JM model will allow us to determine the electrocouplings and the partial πδ, ρp decay widths of several high lying nucleon resonances S31(1620), S11(1650), F15(1685), D33(1700), P13(1720) and to further explore the evidence for the 3/2^+(1720) candidate-state. Analysis of the single pion electroproduction data measured with CLAS in the aforementioned kinematic region is in progress. Single and charged double pion exclusive channels are major contributors to the meson electroproduction in the N* excitation region with different non-resonant mechanisms. A successful description of all observables in these exclusive channels with consistent N* electrocouplings will offer evidence for the reliable evaluation of these fundamental quantities.

  18. CMOS technology and current-feedback op-amps

    DEFF Research Database (Denmark)

    Bruun, Erik

    1993-01-01

    Some of the problems related to the application of CMOS technology to current-feedback operational amplifiers (CFB op-amps) are identified. Problems caused by the low device transconductance and by the absence of matching between p-channel and n-channel transistors are examined, and circuit solut...... poor performance compared to the bipolar designs, but CMOS has a potential for CFB op-amp design if more ingenious circuit configurations are applied...

  19. Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A.

    OpenAIRE

    Bonting, C F; Kortstee, G J; Zehnder, A J

    1991-01-01

    Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity bu...

  20. Bacterial cyclic AMP-phosphodiesterase activity coordinates biofilm formation.

    Directory of Open Access Journals (Sweden)

    Eric J Kalivoda

    Full Text Available Biofilm-related infections are a major contributor to human disease, and the capacity for surface attachment and biofilm formation are key attributes for the pathogenesis of microbes. Serratia marcescens type I fimbriae-dependent biofilms are coordinated by the adenylate cyclase, CyaA, and the cyclic 3',5'-adenosine monophosphate (cAMP-cAMP receptor protein (CRP complex. This study uses S. marcescens as a model system to test the role of cAMP-phosphodiesterase activity in controlling biofilm formation. Herein we describe the characterization of a putative S. marcescens cAMP-phosphodiesterase gene (SMA3506, designated as cpdS, and demonstrated to be a functional cAMP-phosphodiesterase both in vitro and in vivo. Deletion of cpdS resulted in defective biofilm formation and reduced type I fimbriae production, whereas multicopy expression of cpdS conferred a type I fimbriae-dependent hyper-biofilm. Together, these results support a model in which bacterial cAMP-phosphodiesterase activity modulates biofilm formation.

  1. Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

    DEFF Research Database (Denmark)

    Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

    2006-01-01

    In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals...... with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with...

  2. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    Science.gov (United States)

    Antipenko, Y. A.; Bubeyev, Y. A.; Korovkin, B. F.; Mikhaleva, N. P.

    1980-01-01

    Components of the cyclic Adenosine-cyclic-35-monophosphate (AMP) system in the muscle tissue of white rats were studied during 70-75 days of hypokinesia, created by placing the animals in small booths which restricted their movements, and during the readaptation period. In the initial period, cyclic AMP levels and the activities of phosphodiesterase and adenylate cyclase in muscle tissue were increased. The values for these indices were roughly equal for controls and experimental animals during the adaptation period, but on the 70th day of the experiment cAMP levels dropped, phosphodiesterase activity increased, and the stimulative effect of epinephrine on the activity of adenylate cyclase decreased. The indices under study normalized during the readaptation period.

  3. Analysis of AmpC β-lactamase Gene in Pseudomonas aeruginosa

    Institute of Scientific and Technical Information of China (English)

    NI Ming; ZHANG Dongshen; QI Junying

    2005-01-01

    The gene and the amino acid sequence of the structural and regulatory region of the Pseudomonas aeruginosa with different resistance patterns were analyzed. Six strains with different resistance patterns were selected and the AmpC β-lactamase was identified. The objective gene fragment was amplified by colonies PCR. The sequences of the PCR-products were analyzed. The DNA sequence of the structural gene ampC and the regulatory genes ampR, ampD and ampE was detected. The 6 strains and the wild-type Pseudomonas aeruginosa are highly homogeneous in structural and regulatory region. Some new mutant points were found.

  4. Simulation of Organic Solar Cells Using AMPS-1D Program

    Directory of Open Access Journals (Sweden)

    Samah G. Babiker

    2012-03-01

    Full Text Available The analysis of microelectronic and photonic structure in one dimension program [AMPS-1D] program has been successfully used to study inorganic solar cells. In this work the program has been used to optimize the performance of the organic solar cells. The cells considered consist of poly(2-methoxy-5-(3,7- dimethyloctyloxy-1,4-phenylenevinylene [MDMO-PPV

  5. 42 CFR 447.504 - Determination of AMP.

    Science.gov (United States)

    2010-10-01

    ..., dialysis centers, and mental health centers); (9) Sales to mail order pharmacies; (10) Sales to home... Qualified Retiree Prescription Drug Plan under section 1860D-22(a)(2) of the Act, Children's Health... State Medicaid Agencies under section 1927 of the Act. (i) Further clarification of AMP calculation....

  6. Metabolic benefits of inhibiting cAMP-PDEs with resveratrol.

    Science.gov (United States)

    Chung, Jay H

    2012-10-01

    Calorie restriction (CR) extends lifespan in species ranging from yeast to mammals. There is evidence that CR also protects against aging-related diseases in non-human primates. This has led to an intense interest in the development of CR-mimetics to harness the beneficial effects of CR to treat aging-related diseases. One CR-mimetic that has received a great deal of attention is resveratrol. Resveratrol extends the lifespan of obese mice and protects against obesity-related diseases such as type 2 diabetes. The specific mechanism of resveratrol action has been difficult to elucidate because resveratrol has a promiscuous target profile. A recent finding indicates that the metabolic effects of resveratrol may result from competitive inhibition of cAMP-degrading phosphodiesterases (PDEs), which increases cAMP levels. The cAMP-dependent pathways activate AMP-activated protein kinase (AMPK), which is essential for the metabolic effects of resveratrol. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including protection against diet-induced obesity and an increase in mitochondrial function, physical stamina and glucose tolerance in mice. This discovery suggests that PDE inhibitors may be useful for treating metabolic diseases associated with aging. PMID:23700542

  7. Modeling of CO2 absorber using an AMP solution

    DEFF Research Database (Denmark)

    Gabrielsen, Jostein; Michelsen, Michael Locht; Stenby, Erling Halfdan;

    2006-01-01

    Abstract: An explicit model for carbon dioxide (CO2) solubility in an aqueous solution of 2-amino-2-methyl-1-propanol (AMP) has been proposed and an expression for the heat of absorption of CO2 has been developed as a function of loading and temperature. A rate-based steady-state model for CO2...

  8. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  9. Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System.

    Science.gov (United States)

    Zahid, M Shamim Hasan; Awasthi, Sharda Prasad; Asakura, Masahiro; Chatterjee, Shruti; Hinenoya, Atsushi; Faruque, Shah M; Yamasaki, Shinji

    2015-01-01

    Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

  10. The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1985-01-01

    Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (Kd between 15 and 450 nM) and different off-rates of the cAMP-receptor complex (t½ between 0.7 and 150 s). The association of cAMP to the receptor and the

  11. Phosphodiesterase 3 inhibitor cilostazol induces migraine-like attacks via cyclic AMP increase

    DEFF Research Database (Denmark)

    Guo, Song; Olesen, Jes; Ashina, Messoud

    2014-01-01

    The initiating mechanisms of migraine attacks are very complex but may involve the cyclic AMP signalling pathway. It is unknown whether intracellular cyclic AMP accumulation induces migraine attacks. We investigated whether administration of cilostazol, which causes cyclic AMP accumulation, may i...

  12. Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios

    OpenAIRE

    Rui Wu; Meng Zhao; Jing Li; He Gao; Biao Kan; Weili Liang

    2015-01-01

    TfoX (Sxy) and CRP are two important competence activators. The link between tfoX and CRP has been shown in H. influenza but lacking evidence of direct interaction. Recently a Sxy-dependent CRP (CRP-S) site autoregulating Sxy was reported in E. coli. Here, we show that the cAMP-CRP complex transcriptionally regulates tfoX expression through multiple canonical CRP (CRP-N) sites in Vibrios. This conclusion is supported by an analysis of the tfoX mRNA levels and tfoX transcriptional reporter fus...

  13. Design of Low Voltage Low Power CMOS OP-AMP

    Directory of Open Access Journals (Sweden)

    Shahid Khan,

    2014-11-01

    Full Text Available Operational amplifiers are an integral part of many analog and mixed signal systems. As the demand for mixed mode integrated circuits increases, the design of analog circuits such as operational amplifiers in CMOS technology becomes more critical. This paper presents a two stage CMOS operational amplifier, which operates at ±1.8V power supply using TSMC 0.18um CMOS technology. The OP-AMP designed exhibit unity gain frequency of 12.6 MHz, and gain of 55.5db with 300uw power dissipation. The gain margin and phase margin of OP-AMP is 45˚ and 60˚ respectively. Design and simulation has been carried out in P Spice tool.

  14. Redox Regulation of the AMP-Activated Protein Kinase

    OpenAIRE

    Yingying Han; Qilong Wang; Ping Song; Yi Zhu; Ming-Hui Zou

    2010-01-01

    Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death. Objectives The aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC). Methods Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation. Results In BAEC, Berberine caused a dos...

  15. Genetic association of cyclic AMP signaling genes with bipolar disorder

    OpenAIRE

    McDonald, M-L; MacMullen, C.; Liu, D. J.; Leal, S M; Davis, R L

    2012-01-01

    The genetic basis for bipolar disorder (BPD) is complex with the involvement of multiple genes. As it is well established that cyclic adenosine monophosphate (cAMP) signaling regulates behavior, we tested variants in 29 genes that encode components of this signaling pathway for associations with BPD type I (BPD I) and BPD type II (BPD II). A total of 1172 individuals with BPD I, 516 individuals with BPD II and 1728 controls were analyzed. Single SNP (single-nucleotide polymorphism), haplotype...

  16. Software Design Document for the AMP Nuclear Fuel Performance Code

    Energy Technology Data Exchange (ETDEWEB)

    Philip, Bobby [ORNL; Clarno, Kevin T [ORNL; Cochran, Bill [ORNL

    2010-03-01

    The purpose of this document is to describe the design of the AMP nuclear fuel performance code. It provides an overview of the decomposition into separable components, an overview of what those components will do, and the strategic basis for the design. The primary components of a computational physics code include a user interface, physics packages, material properties, mathematics solvers, and computational infrastructure. Some capability from established off-the-shelf (OTS) packages will be leveraged in the development of AMP, but the primary physics components will be entirely new. The material properties required by these physics operators include many highly non-linear properties, which will be replicated from FRAPCON and LIFE where applicable, as well as some computationally-intensive operations, such as gap conductance, which depends upon the plenum pressure. Because there is extensive capability in off-the-shelf leadership class computational solvers, AMP will leverage the Trilinos, PETSc, and SUNDIALS packages. The computational infrastructure includes a build system, mesh database, and other building blocks of a computational physics package. The user interface will be developed through a collaborative effort with the Nuclear Energy Advanced Modeling and Simulation (NEAMS) Capability Transfer program element as much as possible and will be discussed in detail in a future document.

  17. Expression profiles of antimicrobial peptides (AMPs) and their regulation by Relish

    Science.gov (United States)

    Wang, Dongdong; Li, Fuhua; Li, Shihao; Wen, Rong; Xiang, Jianhai

    2012-07-01

    Antimicrobial peptides (AMPs), as key immune effectors, play important roles in the innate immune system of invertebrates. Different types of AMPs, including Penaeidin, Crustin, ALF (antilipopolysaccharide factor) have been identified in different penaeid shrimp; however, systematic analyses on the function of different AMPs in shrimp responsive to different types of bacteria are very limited. In this study, we analyzed the expression profiles of AMPs in the Chinese shrimps, Fenneropenaeus chinensis, simultaneously by real-time RT-PCR (reverse transcription-polymerase chain reaction) when shrimp were challenged with Micrococcus lysodeikticus (Gram-positive, G+) or Vibrio anguillarium (Gram-negative, G-). Different AMPs showed different expression profiles when shrimp were injected with one type of bacterium, and one AMP also showed different expression profiles when shrimp were challenged with different bacteria. Furthermore, the expression of these AMPs showed temporal expression profiles, suggesting that different AMPs function coordinately in bacteria-infected shrimp. An RNA interference approach was used to study the function of the Relish transcription factor in regulating the transcription of different AMPs. The current study showed that Relish could regulate the transcription of different AMPs in shrimp. Differential expression profiles of AMPs in shrimp injected with different types of bacteria indicated that a complicated antimicrobial response network existed in shrimp. These data contribute to our understanding of immunity in shrimp and may provide a strategy for the control of disease in shrimp.

  18. Orthologous and Paralogous AmpD Peptidoglycan Amidases from Gram-Negative Bacteria

    Science.gov (United States)

    Rivera, Ivanna; Molina, Rafael; Lee, Mijoon; Mobashery, Shahriar

    2016-01-01

    Cell wall recycling and β-lactam antibiotic resistance are linked in Enterobacteriaceae and in Pseudomonas aeruginosa. This process involves a large number of murolytic enzymes, among them a cytoplasmic peptidoglycan amidase AmpD, which plays an essential role by cleaving the peptide stem from key intermediates en route to the β-lactamase production (a resistance mechanism) and cell wall recycling. Uniquely, P. aeruginosa has two additional paralogues of AmpD, designated AmpDh2 and AmpDh3, which are periplasmic enzymes. Despite the fact that AmpDh2 and AmpDh3 share a common motif for their respective catalytic domains, they are each comprised of multidomain architectures and exhibit distinct oligomerization properties. We review herein the structural and biochemical properties of orthologous and paralogous AmpD proteins and discuss their implication in cell wall recycling and antibiotic resistance processes. PMID:27326855

  19. Opposing activity changes in AMP deaminase and AMP-activated protein kinase in the hibernating ground squirrel.

    Directory of Open Access Journals (Sweden)

    Miguel A Lanaspa

    Full Text Available Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2 (summer and activation of AMP-activated protein kinase (AMPK (winter. Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2, as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation. Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC and decreased enoyl CoA hydratase (ECH1 and carnitine palmitoyltransferase 1A (CPT1A, rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.

  20. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

    Science.gov (United States)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.

    2016-03-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

  1. Exchange Protein Directly Activated by cAMP (epac) : A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions

    NARCIS (Netherlands)

    Schmidt, Martina; Dekker, Frank J.; Maarsingh, Harm

    2013-01-01

    Since the discovery nearly 60 years ago, cAMP is envisioned as one of the most universal and versatile second messengers. The tremendous feature of cAMP to tightly control highly diverse physiologic processes, including calcium homeostasis, metabolism, secretion, muscle contraction, cell fate, and g

  2. Metabolites of an Epac-selective cAMP analog induce cortisol synthesis by adrenocortical cells through a cAMP-independent pathway.

    Directory of Open Access Journals (Sweden)

    Judith A Enyeart

    Full Text Available Adrenal zona fasciculata (AZF cells express a cAMP-activated guanine nucleotide exchange protein (Epac2 that may function in ACTH-stimulated cortisol synthesis. Experiments were done to determine whether cAMP analogs that selectively activate Epacs could induce cortisol synthesis and the expression of genes coding for steroidogenic proteins in bovine AZF cells. Treatment of AZF cells with the Epac-selective cAMP analog (ESCA 8CPT-2'-OMe-cAMP induced large (>100 fold, concentration-dependent, delayed increases in cortisol synthesis and the expression of mRNAs coding for the steroid hydroxylases CYP11a1, CYP17, CYP21, and the steroid acute regulatory protein (StAR. However, a non-hydrolyzable analog of this ESCA, Sp-8CPT-2'-OMe-cAMP, failed to stimulate cortisol production even at concentrations that activated Rap1, a downstream effector of Epac2. Accordingly, putative metabolites of 8CPT-2'-OMe-cAMP, including 8CPT-2'-OMe-5'AMP, 8CPT-2'-OMe-adenosine, and 8CPT-adenine all induced cortisol synthesis and steroid hydroxylase mRNA expression with a temporal pattern, potency, and effectiveness similar to the parent compound. At concentrations that markedly stimulated cortisol production, none of these metabolites significantly activated cAMP-dependent protein kinase (PKA. These results show that one or more metabolites of the ESCA 8CPT-2'-OMe-cAMP induce cortico-steroidogenesis by activating a panel of genes that code for steroidogenic proteins. The remarkable increases in cortisol synthesis observed in this study appear to be mediated by a novel cAMP-, Epac- and PKA-independent signaling pathway.

  3. Epac and PKA: a tale of two intracellular cAMP receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Cheng; Zhenyu Ji; Tamara Tsalkova; Fang Mei

    2008-01-01

    cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions,including diabetes,heart failure and cancer.In eukaryotic cells,the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors,the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP(Epac)/cAMP-regulated guanine nucleotide exchange factors.Like PKA,Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions.The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner.Depending upon the specific cellular environments as well as their relative abundance,distrbution and localization,Epac and PKA may act independently,converge synergistically or oppose each other in regulating a specific cellular function.

  4. Expression profiles of antimicrobial peptides (AMPs) and their regulation by Relish

    Institute of Scientific and Technical Information of China (English)

    WANG Dongdong; LI Fuhua; LI Shihao; WEN Rong; XIANG Jianhai

    2012-01-01

    Antimicrobial peptides (AMPs),as key immune effectors,play important roles in the innate immune system of invertebrates.Different types of AMPs,including Penaeidin,Crustin,ALF (antilipopolysaccharide factor) have been identified in different penaeid shrimp; however,systematic analyses on the function of different AMPs in shrimp responsive to different types of bacteria are very limited.In this study,we analyzed the expression profiles of AMPs in the Chinese shrimps,Fenneropenaeus chinensis,simultaneously by real-time RT-PCR (reverse transcription-polymerase chain reaction) when shrimp were challenged with Micrococcus lysodeikticus (Gram-positive,G+) or Vibrio anguillarium (Gram-negative,G).Different AMPs showed different expression profiles when shrimp were injected with one type of bacterium,and one AMP also showed different expression profiles when shrimp were challenged with different bacteria.Furthermore,the expression of these AMPs showed temporal expression profiles,suggesting that different AMPs function coordinately in bacteria-infected shrimp.An RNA interference approach was used to study the function of the Relish transcription factor in regulating the transcription of different AM Ps.The current study showed that Relish could regulate the transcription of different AMPs in shrimp.Differential expression profiles of AMPs in shrimp injected with different types of bacteria indicated that a complicated antimicrobial response network existed in shrimp.These data contribute to our understanding of immunity in shrimp and may provide a strategy for the control of disease in shrimp.

  5. DHA-1型质粒AmpC酶调控因子AmpR基因检测%Detection of regulator AmpR genes of DHA-1 type plasmid-encoded AmpC β-lactamase in escherichia coli and klebsiella pnuemoniae isolates

    Institute of Scientific and Technical Information of China (English)

    多丽波; 栾英; 张联博; 李垚

    2007-01-01

    目的 检测DHA-1型质粒AmpC酶诱导性耐药调控因子AmpR基因.方法 以我院临床分离产DHA-1型质粒AmpC酶的4株大肠埃希菌和10株肺炎克雷伯菌为研究对象,采用聚合酶链反应(PCR),用特异性引物扩增AmpR调节因子全编码基因,并将一株肺炎克雷伯菌AmpR基因克隆到pET-22b(+)载体质粒中测序.结果 所有实验菌株AmpR基因检测阳性,重组子AmpR基因测序表明与摩根摩根菌染色体上AmpR基因同源性达99.8%.结论 产DHA-1型质粒AmpC酶肺炎克雷伯菌、大肠埃希菌的质粒上存在调控因子AmpR基因.

  6. Imaging cyclic AMP changes in pancreatic islets of transgenic reporter mice.

    Directory of Open Access Journals (Sweden)

    Joung Woul Kim

    Full Text Available Cyclic AMP (cAMP and Ca(2+ are two ubiquitous second messengers in transduction pathways downstream of receptors for hormones, neurotransmitters and local signals. The availability of fluorescent Ca(2+ reporter dyes that are easily introduced into cells and tissues has facilitated analysis of the dynamics and spatial patterns for Ca(2+ signaling pathways. A similar dissection of the role of cAMP has lagged because indicator dyes do not exist. Genetically encoded reporters for cAMP are available but they must be introduced by transient transfection in cell culture, which limits their utility. We report here that we have produced a strain of transgenic mice in which an enhanced cAMP reporter is integrated in the genome and can be expressed in any targeted tissue and with tetracycline induction. We have expressed the cAMP reporter in beta-cells of pancreatic islets and conducted an analysis of intracellular cAMP levels in relation to glucose stimulation, Ca(2+ levels, and membrane depolarization. Pancreatic function in transgenic mice was normal. In induced transgenic islets, glucose evoked an increase in cAMP in beta-cells in a dose-dependent manner. The cAMP response is independent of (in fact, precedes the Ca(2+ influx that results from glucose stimulation of islets. Glucose-evoked cAMP responses are synchronous in cells throughout the islet and occur in 2 phases suggestive of the time course of insulin secretion. Insofar as cAMP in islets is known to potentiate insulin secretion, the novel transgenic mouse model will for the first time permit detailed analyses of cAMP signals in beta-cells within islets, i.e. in their native physiological context. Reporter expression in other tissues (such as the heart where cAMP plays a critical regulatory role, will permit novel biomedical approaches.

  7. Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP.

    Science.gov (United States)

    Sucic, J F; Selmin, O; Rutherford, C L

    1993-01-01

    A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely. PMID:8222346

  8. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Directory of Open Access Journals (Sweden)

    Pei-Chi Yang

    2016-07-01

    Full Text Available Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and

  9. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Science.gov (United States)

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal

  10. Nanomolar Inhibitors of AmpC [beta]-Lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Morandi, Federica; Caselli, Emilia; Morandi, Stefania; Focia, Pamela J.; Blazquez, Jesus; Shoichet, Brian K.; Prati, Fabio (Degali); (NIH); (NWU); (UCSF)

    2010-03-08

    {beta}-lactamases are the most widespread resistance mechanism to {beta}-lactam antibiotics, such as the penicillins and the cephalosporins. In an effort to combat these enzymes, a combination of stereoselective organic synthesis, enzymology, microbiology, and X-ray crystallography was used to design and evaluate new carboxyphenyl-glycylboronic acid transition-state analogue inhibitors of the class C {beta}-lactamase AmpC. The new compounds improve inhibition by over 2 orders of magnitude compared to analogous glycylboronic acids, with K{sub i} values as low as 1 nM. On the basis of the differential binding of different analogues, the introduced carboxylate alone contributes about 2.1 kcal/mol in affinity. This carboxylate corresponds to the ubiquitous C3(4)' carboxylate of {beta}-lactams, and this energy represents the first thermodynamic measurement of the importance of this group in molecular recognition by class C {beta}-lactamases. The structures of AmpC in complex with two of these inhibitors were determined by X-ray crystallography at 1.72 and 1.83 {angstrom} resolution. These structures suggest a structural basis for the high affinity of the new compounds and provide templates for further design. The highest affinity inhibitor was 5 orders of magnitude more selective for AmpC than for characteristic serine proteases, such as chymotrypsin. This inhibitor reversed the resistance of clinical pathogens to the third generation cephalosporin ceftazidime; it may serve as a lead compound for drug discovery to combat bacterial resistance to {beta}-lactam antibiotics.

  11. The AMP (Advanced MultiPhysics) Nuclear Fuel Performance code

    International Nuclear Information System (INIS)

    Highlights: ► New, three-dimensional, parallel, multi-physics code to simulate fuel behavior in nominal operation. ► Fully-coupled thermomechanics for nominal operation and operation during transients. ► Isotopic depletion using Scale/ORIGEN-S within a fuel performance code. ► Leveraging of existing, validated material models from existing fuel performance codes. ► Initial validation evaluation of an advanced modeling and simulation code for fuel performance. - Abstract: The AMP (Advanced MultiPhysics) Nuclear Fuel Performance code is a new, three-dimensional, multi-physics tool that uses state-of-the-art solution methods and validated nuclear fuel models to simulate the nominal operation and anticipated operational transients of nuclear fuel. The AMP Nuclear Fuel Performance code leverages existing validated material models from traditional fuel performance codes and the Scale/ORIGEN-S spent-fuel characterization code to provide an initial capability that is shown to be sufficiently accurate for a single benchmark problem and anticipated to be accurate for a broad range of problems. The thermomechanics foundation can be solved in a time-dependent or quasi-static approach with any variation of operator-split or fully-coupled solutions at each time step through interoperable interfaces to leading computational mathematics tools, including PETSc, Trilinos, and SUNDIALS. A baseline validation of the AMP Nuclear Fuel Performance code has been performed through the modeling of an experiment in the Halden Reactor Project (IFA-432) that demonstrates the integrated capability and provides a baseline of the initial accuracy of the software.

  12. Op. amps in power amplification: a laboratory exercise on feedback

    Science.gov (United States)

    Borcherds, P. H.

    1984-05-01

    Rapid and continuing developments in electronics make it necessary to revise constantly the teaching of electronics and to replace obsolescent laboratory exercises. The author describes a new exercise which believes helps students' (and lecturers') understanding of negative feedback. A power amplifier is constructed from an operational amplifier (op. amp.) together with a complementary pair of transistors as an output stage. To make the exercise more realistic a low impedance (8 Omega ) loudspeaker is used as the load. The amplifier is developed and tested stage by stage, and at each stage the defects apparent at the previous stage are eliminated.

  13. Buddhism in Čampā Le Bouddhisme au Champa

    Directory of Open Access Journals (Sweden)

    Anne-Valérie Schweyer

    2009-10-01

    Full Text Available Čampā is a Far East country, whose Māhāyana Buddhism is known from 7th to 14th century. In fact, Sanskrit and Cam Inscriptions mostly attested Tantric practices, belonging to the Vajrāyana Buddhism, mixing Śaiva and Buddhist believes. More precisely, side by side Śiva and the three Buddha’ emanations, Śākyamuni, Amitābha and Vairocana, are honoured in Čampā, alone with the Goddess Prajñāpāramitā, the true substance of the Doctrine, and, secondary, with Vajrapāni, Lokeśvara and Vajrasattva. The confrontation of the epigraphic testimonies with the archaeological remains is very useful to understand the Buddhism of Čampā, crossroads of trade roads between India and China. Therefore, epigraphic and artistic evidences are used to propose a chronological presentation, with a special development on the revival of the 10th century, and especially, the esoteric way.Le Čampā, pays du centre Vietnam, connut un bouddhisme Māhāyana du xe au xive siècle. Les inscriptions en sanskrit et en cam montrent que ce bouddhisme était essentiellement tantrique, relevant du bouddhisme Vajrāyana, mêlant pratiques Śivaïtes et bouddhiques. Plus précisément, le bouddhisme cam montre qu’aux côtés de Śiva sont honorées les trois émanations du Bouddha, Śākyamuni, Amitābha et Vairocana, avec la déesse Prajñāpāramitā, la Vraie Substance de la Connaissance ; on trouve également Vajrapāni, Lokeśvara et Vajrasattva. La confrontation des témoignages épigraphiques et archéologiques permet de mieux appréhender le bouddhisme cam, à la croisée des routes commerciales entre l’Inde et la Chine. Cet article exploite ces témoignages dans une perspective chronologique, avec un développement particulier pour le Bouddhisme ésotérique au xe siècle.

  14. Cyclic AMP enhances TGFβ responses of breast cancer cells by upregulating TGFβ receptor I expression.

    Directory of Open Access Journals (Sweden)

    Ilka Oerlecke

    Full Text Available Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein and TβRI (TGFβ receptor 1, were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation

  15. Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani.

    Science.gov (United States)

    Jha, Sanjay; Tank, Harsukh G; Prasad, Bishun Deo; Chattoo, Bharat B

    2009-02-01

    Magnaporthe oryzae and Rhizoctonia solani, are among the most important pathogens of rice, severely limiting its productivity. Dm-AMP1, an antifungal plant defensin from Dahlia merckii, was expressed in rice (Oryza sativa L. sp. indica cv. Pusa basmati 1) using Agrobacterium tumefaciens-mediated transformation. Expression levels of Dm-AMP1 ranged from 0.43% to 0.57% of total soluble protein in transgenic plants. It was observed that constitutive expression of Dm-AMP1 suppresses the growth of M. oryzae and R. solani by 84% and 72%, respectively. Transgenic expression of Dm-AMP1 was not accompanied by an induction of pathogenesis-related (PR) gene expression, indicating that the expression of DmAMP1 directly inhibits the pathogen. The results of in vitro, in planta and microscopic analyses suggest that Dm-AMP1 expression has the potential to provide broad-spectrum disease resistance in rice. PMID:18618285

  16. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    Science.gov (United States)

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  17. Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy.

    Science.gov (United States)

    Yang, Haihua; Yang, Linghai

    2016-08-01

    In mammals, cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is usually elicited by binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs). cAMP exerts many of its physiological effects by activating cAMP-dependent protein kinase (PKA), which in turn phosphorylates and regulates the functions of downstream protein targets including ion channels, enzymes, and transcription factors. cAMP/PKA signaling pathway regulates glucose homeostasis at multiple levels including insulin and glucagon secretion, glucose uptake, glycogen synthesis and breakdown, gluconeogenesis, and neural control of glucose homeostasis. This review summarizes recent genetic and pharmacological studies concerning the regulation of glucose homeostasis by cAMP/PKA in pancreas, liver, skeletal muscle, adipose tissues, and brain. We also discuss the strategies for targeting cAMP/PKA pathway for research and potential therapeutic treatment of type 2 diabetes mellitus (T2D). PMID:27194812

  18. Mathematical model of cAMP-dependent signaling pathway in constitutive and UV-induced melanogenesis

    Science.gov (United States)

    Stolnitz, Mikhail M.; Peshkova, Anna Y.

    2002-07-01

    Cascade of reactions of cAMP-dependent signaling pathway in melanocytes is investigated by mathematical modeling. Model takes into account (alpha) -melanocyte stimulating hormone binding to melanocortin-1 receptor, adenylate cyclase activation by G-protein, increase of the intracellular cAMP concentration, PKA activation by cAMP, CREB phosphorylation by PKA, microphthalmia gene expression, microphthalmia binding to tyrosinase gene promoter, increase of tyrosinase synthesis. Positive and negative feedback loops of this system are analyzed.

  19. cAMP diffusion in Dictyostelium discoideum: A Green's function method

    Science.gov (United States)

    Calovi, Daniel S.; Brunnet, Leonardo G.; de Almeida, Rita M. C.

    2010-07-01

    A Green’s function method is developed to approach the spatiotemporal equations describing the cAMP production in Dictyostelium discoideum, markedly reducing numerical calculations times: cAMP concentrations and gradients are calculated just at the amoeba locations. A single set of parameters is capable of reproducing the different observed behaviors, from cAMP synchronization, spiral waves and reaction-diffusion patterns to streaming and mound formation. After aggregation, the emergence of a circular motion of amoebas, breaking the radial cAMP field symmetry, is observed.

  20. Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR.

    Science.gov (United States)

    Caille, Olivier; Zincke, Diansy; Merighi, Massimo; Balasubramanian, Deepak; Kumari, Hansi; Kong, Kok-Fai; Silva-Herzog, Eugenia; Narasimhan, Giri; Schneper, Lisa; Lory, Stephen; Mathee, Kalai

    2014-11-01

    Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC β-lactamase is a common mechanism of β-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (PampC) and ampR (PampR) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5' rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ(54) and σ(70) consensus sequences were identified at PampR and PampC, respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal PampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to PampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding. PMID:25182487

  1. Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

    Science.gov (United States)

    Krishnamurthy, Srinath; Moorthy, Balakrishnan Shenbaga; Xin Xiang, Lim; Xin Shan, Lim; Bharatham, Kavitha; Tulsian, Nikhil Kumar; Mihalek, Ivana; Anand, Ganesh S

    2014-09-16

    Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and

  2. Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases

    DEFF Research Database (Denmark)

    Friis, Ulla G; Jensen, Boye L; Sethi, Shala;

    2002-01-01

    , and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol...... trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed....../L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects...

  3. Coronary vasodilatory, spasmolytic and cAMP-phosphodiesterase inhibitory properties of dihydropyranocoumarins and dihydrofuranocoumarins

    DEFF Research Database (Denmark)

    Thastrup, Ole; Fjalland, B; Lemmich, J

    1983-01-01

    Twenty-three dihydropyrano- and dihydrofuranocoumarins, most of plant origin, were examined for their effects on the coronary flow of isolated perfused guinea-pig heart, on the Ba2+-induced spasms in isolated guinea-pig ileum, on the cAMP level in guinea-pig heart homogenate and on the cAMP metab...... between the coronary vasodilatory and the cAMP-phosphodiesterase inhibitory activity. The results indicate involvement of cAMP-phosphodiesterase inhibition in coronary vasodilatory effects of acyloxydihydropyrano- and acyloxydihydrofurano-coumarins....

  4. 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.

  5. Extracellular ATP and Dibutyryl cAMP Enhance the Freezability of Rat Epididymal Sperm

    OpenAIRE

    Yamashiro, Hideaki; Toyomizu, Masaaki; Toyama, Natsuki; Aono, Nobuya; Sakurai, Masahiro; HIRADATE, Yuuki; Yokoo, Masaki; Moisyadi, Stefan; Sato, Eimei

    2010-01-01

    We studied the effects of ATP, ionomycin, and dibutyryl cAMP (dbcAMP) on the motility, freezability, and oxygen consumption of rat epididymal sperm. In vitro fertilization and intrauterine insemination were performed by using frozen–thawed rat sperm. Frozen–thawed sperm diluted in raffinose–modified Krebs-Ringer bicarbonate solution–egg yolk extender containing 1.85 mM ATP and 100 μM dbcAMP exhibited considerably higher motility and viability than sperm diluted in dbcAMP-free extender. Additi...

  6. TSH-induced cyclic AMP production in an ovine thyroid cell line: OVNIS 5H.

    Science.gov (United States)

    Fayet, G; Aouani, A; Hovsépian, S

    1986-01-01

    The TSH-induced cyclic AMP response was studied using a 3-year-old ovine thyroid cell line TSH-independent for growth: OVNIS 5H. The kinetics of cyclic AMP production was followed both in cell layers and in cell culture media, with or without phosphodiesterase inhibitor. It is noteworthy that following the first wave in cyclic AMP obtained within minutes, we observed later a sustained exponential increase in cyclic AMP during the 5 days following TSH stimulation. A bioassay of TSH was derived allowing measurement of 1 microU/ml TSH from a crude bTSH preparation. PMID:3000830

  7. Genetically-encoded tools for cAMP probing and modulation in living systems.

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov

    2015-09-01

    Full Text Available Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming - all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells, underpin the ensuing limitations of the conventional cAMP assays: 1 genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; 2 inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control – something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.

  8. Heterogeneity of Calcium Channel/cAMP-Dependent Transcriptional Activation.

    Science.gov (United States)

    Kobrinsky, Evgeny

    2015-01-01

    The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

  9. Elevated cAMP increases aquaporin-3 plasma membrane diffusion

    DEFF Research Database (Denmark)

    Marlar, Saw; Christensen, Eva Arnspang; Koffman, Jennifer Skaarup;

    2014-01-01

    .05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption......Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water...... be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal...

  10. Amp: A modular approach to machine learning in atomistic simulations

    Science.gov (United States)

    Khorshidi, Alireza; Peterson, Andrew A.

    2016-10-01

    Electronic structure calculations, such as those employing Kohn-Sham density functional theory or ab initio wavefunction theories, have allowed for atomistic-level understandings of a wide variety of phenomena and properties of matter at small scales. However, the computational cost of electronic structure methods drastically increases with length and time scales, which makes these methods difficult for long time-scale molecular dynamics simulations or large-sized systems. Machine-learning techniques can provide accurate potentials that can match the quality of electronic structure calculations, provided sufficient training data. These potentials can then be used to rapidly simulate large and long time-scale phenomena at similar quality to the parent electronic structure approach. Machine-learning potentials usually take a bias-free mathematical form and can be readily developed for a wide variety of systems. Electronic structure calculations have favorable properties-namely that they are noiseless and targeted training data can be produced on-demand-that make them particularly well-suited for machine learning. This paper discusses our modular approach to atomistic machine learning through the development of the open-source Atomistic Machine-learning Package (Amp), which allows for representations of both the total and atom-centered potential energy surface, in both periodic and non-periodic systems. Potentials developed through the atom-centered approach are simultaneously applicable for systems with various sizes. Interpolation can be enhanced by introducing custom descriptors of the local environment. We demonstrate this in the current work for Gaussian-type, bispectrum, and Zernike-type descriptors. Amp has an intuitive and modular structure with an interface through the python scripting language yet has parallelizable fortran components for demanding tasks; it is designed to integrate closely with the widely used Atomic Simulation Environment (ASE), which

  11. Cyclic AMP suppresses interleukin-5 synthesis by human helper T cells via the downregulation of the calcium mobilization pathway

    OpenAIRE

    Kaminuma, Osamu; Mori, Akio; Ogawa, Koji; Kikkawa, Hideo; Nakata, Aya; Ikezawa, Katsuo; Okudaira, Hirokazu

    1999-01-01

    To delineate the mechanism by which cyclic AMP (cAMP) suppresses interleukin (IL)-5 synthesis, the effects of prostaglandin (PG) E2, forskolin, dibutyryl (db)-cAMP and the Ca2+ ionophore, ionomycin on cytokine synthesis, proliferation and CD25 expression of human T cells were investigated. Further studies were performed by measurement of the intracellular concentrations of cyclic AMP ([cAMP]i) and Ca2+ ([Ca2+]i) and by electrophoretic mobility shift analysis (EMSA).PGE2, forskolin and db-cAMP...

  12. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated transm

  13. Targeting brain tumor cAMP: the case for sex-specific therapeutics

    Directory of Open Access Journals (Sweden)

    Nicole M Warrington

    2015-07-01

    Full Text Available A relationship between cyclic adenosine 3’, 5’-monophosphate (cAMP levels and brain tumor biology has been evident for nearly as long as cAMP and its synthetase, adenylate cyclase (ADCY have been known. The importance of the pathway in brain tumorigenesis has been demonstrated in vitro and in multiple animal models. Recently, we provided human validation for a cooperating oncogenic role for cAMP in brain tumorigenesis when we found that SNPs in ADCY8 were correlated with glioma (brain tumor risk in individuals with Neurofibromatosis type 1 (NF1. Together, these studies provide a strong rationale for targeting cAMP in brain tumor therapy. However, the cAMP pathway is well known to be sexually dimorphic, and SNPs in ADCY8 affected glioma risk in a sex-specific fashion, elevating the risk for females while protecting males. The cAMP pathway can be targeted at multiple levels in the regulation of its synthesis and degradation. Sex differences in response to drugs that target cAMP regulators indicate that successful targeting of the cAMP pathway for brain tumor patients is likely to require matching specific mechanisms of drug action with patient sex.

  14. cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors

    DEFF Research Database (Denmark)

    Mathiesen, Jesper Mosolff; Vedel, Line; Bräuner-Osborne, Hans

    2013-01-01

    Cyclic adenosine monophosphate (cAMP) is a common second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of G(s)-coupled G protein-coupled receptors (GPCRs) and decreased by activation of G(i)-coupled GPCRs via the adenylyl cyclase. Man...

  15. Design of cAMP-CRP-activated promoters in Escherichia coli

    DEFF Research Database (Denmark)

    Valentin-Hansen, P; Holst, B; Søgaard-Andersen, L;

    1991-01-01

    We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10...

  16. A Temporal-Specific and Transient cAMP Increase Characterizes Odorant Classical Conditioning

    Science.gov (United States)

    Cui, Wen; Smith, Andrew; Darby-King, Andrea; Harley, Carolyn W.; McLean, John H.

    2007-01-01

    Increases in cyclic adenosine monophosphate (cAMP) are proposed to initiate learning in a wide variety of species. Here, we measure changes in cAMP in the olfactory bulb prior to, during, and following a classically conditioned odor preference trial in rat pups. Measurements were taken up to the point of maximal CREB phosphorylation in olfactory…

  17. The cAMP Signaling and MAP Kinase Pathways in Plant Pathogenic Fungi

    NARCIS (Netherlands)

    Mehrabi, R.; Zhao, X.; Kim, Y.; Xu, J.R.

    2009-01-01

    The key components of the well conserved cyclic AMP signaling and MAP kinase pathways have been functionally characterized in the corn smut Ustilago maydis, rice blast fungus Magnaporthe grisea, and a few other fungal pathogens. In general, the cAMP signaling and the MAP kinase cascade homologous to

  18. cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids

    NARCIS (Netherlands)

    Madsen, L.; Pedersen, L.M.; Liaset, B.; Ma, T.; Petersen, R.K.; Berg, S. van den; Pan, J.; Müller-Decker, K.M.; Dülsner, E.D.; Kleemann, R.; Kooistra, T.; Døskeland, S.O.; Kristiansen, K.

    2008-01-01

    The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti-ad

  19. BaAMPs: the database of biofilm-active antimicrobial peptides.

    Science.gov (United States)

    Di Luca, Mariagrazia; Maccari, Giuseppe; Maisetta, Giuseppantonio; Batoni, Giovanna

    2015-01-01

    Antimicrobial peptides (AMPs) are increasingly being considered as novel agents against biofilms. The development of AMP-based anti-biofilm strategies strongly relies on the design of sequences optimized to target specific features of sessile bacterial/fungal communities. Although several AMP databases have been created and successfully exploited for AMP design, all of these use data collected on peptides tested against planktonic microorganisms. Here, an open-access, manually curated database of AMPs specifically assayed against microbial biofilms (BaAMPs) is presented for the first time. In collecting relevant data from the literature an effort was made to define a minimal standard set of essential information including, for each AMP, the microbial species and biofilm conditions against which it was tested, and the specific assay and peptide concentration used. The availability of these data in an organized framework will benefit anti-biofilm research and support the design of novel molecules active against biofilm. BaAMPs is accessible at http://www.baamps.it. PMID:25760404

  20. Functional involvement of Annexin-2 in cAMP induced AQP2 trafficking.

    NARCIS (Netherlands)

    Tamma, G.; Procino, G.; Mola, M.G.; Svelto, M.; Valenti, G.

    2008-01-01

    Annexin-2 is required for the apical transport in epithelial cells. In this study, we investigated the involvement of annexin-2 in cAMP-induced aquaporin-2 (AQP2) translocation to the apical membrane in renal cells. We found that the cAMP-elevating agent forskolin increased annexin-2 abundance in th

  1. The effect of neuropeptides on vessel tone and cAMP production

    DEFF Research Database (Denmark)

    Yao, W; Sheikh, S P; Ottesen, B;

    1996-01-01

    The effect of VIP, PHM, PHV, PACAP-27, and PACAP-38 on vessel tone and cAMP production was investigated in rabbit ovarian arteries in vitro. The peptides (10(-7)M) induced a significant relaxation on NA-precontracted vessels and displayed similar potencies. The cAMP accumulation induced by PACAP-...

  2. Model development and process simulation of postcombustion carbon capture technology with aqueous AMP/PZ solvent

    NARCIS (Netherlands)

    van der Spek, Mijndert; Arendsen, Richard; Ramirez, Andrea; Faaij, André

    2016-01-01

    This study presents the development, application, and uncertainty analysis of a process simulation model for postcombustion CO2 capture with an AMP/PZ solvent blend based on state of the art knowledge on AMP/PZ solvent technology. The development includes the improvement of the physical property mod

  3. 78 FR 1264 - CalAmp Wireless Networks Corporation, Waseca, MN; Notice of Negative Determination Regarding...

    Science.gov (United States)

    2013-01-08

    ... Employment and Training Administration CalAmp Wireless Networks Corporation, Waseca, MN; Notice of Negative... workers of the subject firm (TA-W-80,399A; CalAmp Wireless Networks Corporation, Waseca, Minnesota... Wireless Networks Corporation, Waseca, Minnesota to apply for TAA, the Department determines that...

  4. Global and local missions of cAMP signaling in neural plasticity, learning and memory

    Directory of Open Access Journals (Sweden)

    Daewoo eLee

    2015-08-01

    Full Text Available The fruit fly Drosophila melanogaster has been a popular model to study cAMP signaling and resultant behaviors due to its powerful genetic approaches. All molecular components (AC, PDE, PKA, CREB, etc essential for cAMP signaling have been identified in the fly. Among them, adenylyl cyclase (AC gene rutabaga and phosphodiesterase (PDE gene dunce have been intensively studied to understand the role of cAMP signaling. Interestingly, these two mutant genes were originally identified on the basis of associative learning deficits. This commentary summarizes findings on the role of cAMP in Drosophila neuronal excitability, synaptic plasticity and memory. It mainly focuses on two distinct mechanisms (global versus local regulating excitatory and inhibitory synaptic plasticity related to cAMP homeostasis. This dual regulatory role of cAMP is to increase the strength of excitatory neural circuits on one hand, but to act locally on postsynaptic GABA receptors to decrease inhibitory synaptic plasticity on the other. Thus the action of cAMP could result in a global increase in the neural circuit excitability and memory. Implications of this cAMP signaling related to drug discovery for neural diseases are also described.

  5. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  6. Role of AC-cAMP-PKA Cascade in Antidepressant Action of Electroacupuncture Treatment in Rats

    Directory of Open Access Journals (Sweden)

    Jian-hua Liu

    2012-01-01

    Full Text Available Adenylyl cyclase (AC-cyclic adenosine monophosphate (cAMP-cAMP-dependent protein kinase A (PKA cascade is considered to be associated with the pathogenesis and treatment of depression. The present study was conducted to explore the role of the cAMP cascade in antidepressant action of electroacupuncture (EA treatment for chronic mild stress (CMS-induced depression model rats. The results showed that EA improved significantly behavior symptoms in depression and dysfunction of AC-cAMP-PKA signal transduction pathway induced by CMS, which was as effective as fluoxetine. Moreover, the antidepressant effects of EA rather than Fluoxetine were completely abolished by H89, a specific PKA inhibitor. Consequently, EA has a significant antidepressant treatment in CMS-induced depression model rats, and AC-cAMP-PKA signal transduction pathway is crucial for it.

  7. Structural Elucidation and Antitumor Activity of Polysaccharide AMP-1 from Atractylodis macrocephalae K.

    Institute of Scientific and Technical Information of China (English)

    SHAN,Jun-Jie; KE,Wei; DENG,Jun-E; TIAN,Geng-Yuan

    2003-01-01

    A polysaccharide named as AMP-1 was isolated from the root ofAtractylodis macrocephalae Koidz, and purified by ethanol fractionation and gel filtration. The homogeneity of AMP-1 was determined by HPLC and capillary electrophoresis that gave a single peak.AMP-1 is composed of galactose and mannose in a molar ratio of 1.0:1.9. Its molecular weight is3.8× 103. The structure of glycan was elucidated by IR, 1H NMR, 13C NMR and methylation analysis, and on the basis of the results was suggested that AMP-1 contain a backbone of β- (1→2)-D-galactose residues with branches of single β-D-mannose residue being substituted at the 0-6. Bioactivity assay of AMP-1 showed that it could inhibit growth of Sarcoma 180 and Lewis pulmonary carcinoma implanted in mice.

  8. Kinetics study of CO2 absorption in AMP and Piperazine solutions

    OpenAIRE

    Usman, Muhammad

    2012-01-01

    Kinetics of CO2 in AMP (2-amino 2-methyl 1-propanol) with concentration of 0.1/0.5/1.0/2.0/3.0/4.0 M, 3M AMP with CO2 loadings of 0.15/0.22/0.29 and 0.1/0.5/1.0/1.5M piperazine solutions were measured at a temperature range of 25-70oC. The AMP system was measured at 1KPa pressure of CO2 while CO2- loaded AMP and piperazine were measured at different partial pressures that range from 1-9KPa. The experiments for AMP system were performed in string of disc contactor while PZ system was measured ...

  9. AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo

    Science.gov (United States)

    Dai, Tongcheng; Li, Na; Liu, Lu; Liu, Qin; Zhang, Yuanxing

    2015-11-01

    Quantum dots (QDs) are engineered nanoparticles that possess special optical and electronic properties and have shown great promise for future biomedical applications. In this work, adenosine 5'-monophosphate (AMP), a small biocompatible molecular, was conjugated to organic QDs to produce hydrophilic AMP-QDs. Using macrophage J774A.1 as the cell model, AMP-QDs exhibited both prior imaging property and low toxicity, and more importantly, triggered limited innate immune responses in macrophage, indicating low immunotoxicity in vitro. Using BALB/c mice as the animal model, AMP-QDs were found to be detained in immune organs but did not evoke robust inflammation responses or obvious histopathological abnormalities, which reveals low immunotoxicity in vivo. This work suggests that AMP is an excellent surface ligand with low immunotoxicity, and potentially used in surface modification for more extensive nanoparticles.

  10. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

    Science.gov (United States)

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  11. cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models.

    Science.gov (United States)

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; van de Klundert, Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; van Blitterswijk, Clemens; de Boer, Jan

    2009-08-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications. PMID:19231969

  12. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    Energy Technology Data Exchange (ETDEWEB)

    Gapstur, S.M.; Homma, S.; Dousa, T.P.

    1988-08-01

    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-(32P)-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-(32P)cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-(32P)cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.

  13. 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.

  14. Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4

    Energy Technology Data Exchange (ETDEWEB)

    Kucka, Marek [Section on Cellular Signaling, Program in Developmental Neuroscience, NICHD, NIH, Bethesda, MD (United States); Pogrmic-Majkic, Kristina; Fa, Svetlana [Laboratory for Ecotoxicology, Department of Biology and Ecology, University of Novi Sad, Faculty of Sciences, 21000 Novi Sad (Serbia); Stojilkovic, Stanko S. [Section on Cellular Signaling, Program in Developmental Neuroscience, NICHD, NIH, Bethesda, MD (United States); Kovacevic, Radmila, E-mail: radmila.kovacevic@dbe.uns.ac.rs [Laboratory for Ecotoxicology, Department of Biology and Ecology, University of Novi Sad, Faculty of Sciences, 21000 Novi Sad (Serbia)

    2012-11-15

    Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s. -- Highlights: ► Atrazine stimulates cAMP accumulation in pituitary and Leydig cells. ► Atrazine also stimulates PRL and androgens secretion. ► Stimulatory effects of atrazine were abolished in cells with IBMX-inhibited PDEs. ► Atrazine specificity toward cAMP

  15. Is the level of cyclic AMP in the pyloric caeca of the starfish Asterias rubens related to the reproductive cycle?

    NARCIS (Netherlands)

    Voogt, P.A.; Rheenen, J.W.A. van

    1984-01-01

    1. 1. Cyclic AMP levels were significantly higher (P = 0.01) in female than in male animals. The average difference found was 0.50 pmole/mg dry weight. 2. 2. Cyclic AMP levels were negatively correlated to the pyloric caeca-index. In females this correlation was −0.54 (P < 0.05). 3. 3. Cyclic AMP

  16. Cyclic AMP represents a crucial component of Treg cell-mediated immune regulation

    Directory of Open Access Journals (Sweden)

    Tobias Bopp

    2016-08-01

    Full Text Available T regulatory (Treg cells are one of the key players in the immune tolerance network (ITN and a plethora of manuscripts has described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted the source and the mechanism of action of cAMP are less clear and a multitude of seemingly contradictory data allow for in principle two different scenarios of cAMP-mediated suppression. In one scenario Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication (GJIC directly to the effector T cells (Teff leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine which trigger the adenylate cyclases (AC in Teff via A2A and A2B receptors thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation.

  17. Dopamine-induced cyclic AMP increase in canine myocardium, kidney and superior mesenteric artery.

    Directory of Open Access Journals (Sweden)

    Kazuno,Hiroshi

    1982-04-01

    Full Text Available The effect of dopamine on cyclic AMP levels in tissue slices of canine myocardium and kidney, and in chopped superior mesenteric arterial wall was investigated to identify dopamine receptors. Tissues were incubated in modified Krebs-Henseleit Ringer bicarbonate solution at 37 degrees C for 20 min with test drugs, after 20-min preincubation. In the presence of 3-isobutyl-1-methylxanthine (IBMX, dopamine and apomorphine caused dose-dependent increases in cyclic AMP levels in the myocardium, kidney and superior mesenteric artery. Phentolamine significantly intensified the cyclic AMP-increasing effect of dopamine in the superior mesenteric artery, but it did not influence the cyclic AMP increase caused by dopamine or apomorphine in the myocardium and kidney. Propranolol markedly blocked the effect of dopamine on cyclic AMP levels in all tissues studied. Haloperidol slightly inhibited the effect of dopamine and completely blocked the effect of apomorphine in the myocardium and kidney. These data suggest that dopamine increases cyclic AMP levels by activating predominantly beta-adrenergic receptors and partly dopamine receptors in the canine myocardium, kidney and superior mesenteric artery. The present results also suggest that dopamine acts not only on beta-adrenergic and dopamine receptors but also on alpha-adrenergic receptors in the superior mesenteric artery. Contrary to the activation of beta-adrenergic and dopamine receptors, the activation of alpha-adrenergic receptors resulted in a decrease in cyclic AMP levels in this tissue.

  18. The effect of radioprotectors and ionizing radiation on the cAMP system

    International Nuclear Information System (INIS)

    A number of changes characteristic of all the radioprotectors which appear to play a key role in the radioprotective effect can be distinguished from a variety of radioprotector-caused processes in cells and tissues known to date. Experiments have revealed an increase in cAMP concentration in cells and tissues under the effect of radioprotectors belonging to different classes of chemical compounds. Biogenic amines account for the following reaction sequence: adenylate cyclase activation followed by an increase in cAMP concentration. The augmentation of cAMP level resultant from the effect of sulfur-containing protectors takes an indirect route since adenylate cyclase activation or phosphodiesterase inhibition have not been recorded. It is suggested that an increase in cAMP concentration brought about by sulfur-containing protectors is mediated by biogenic amines as these protectors activate biogenic amine synthesis to increase the level of biogenic amines in tissues, biogenic amines in these concentrations activating adenylate cyclase. The evidence on the dynamics of cAMP level changes after administration of radioprotectors are consistent with the above suggestion. Investigations of the effect of ionizing radiations on the intracellular regulator (cAMP) system account for some of the manifestations of radiation disease. Some data on the effect of radiations on the cAMP system enzymes are given. Changes in the enzyme activity are noted. Possible mechanisms and consequences of these changes are discussed

  19. Second Messenger Signaling in Bacillus subtilis: Accumulation of Cyclic di-AMP Inhibits Biofilm Formation.

    Science.gov (United States)

    Gundlach, Jan; Rath, Hermann; Herzberg, Christina; Mäder, Ulrike; Stülke, Jörg

    2016-01-01

    The Gram-positive model organism Bacillus subtilis produces the essential second messenger signaling nucleotide cyclic di-AMP. In B. subtilis and other bacteria, c-di-AMP has been implicated in diverse functions such as control of metabolism, cell division and cell wall synthesis, and potassium transport. To enhance our understanding of the multiple functions of this second messenger, we have studied the consequences of c-di-AMP accumulation at a global level by a transcriptome analysis. C-di-AMP accumulation affected the expression of about 700 genes, among them the two major operons required for biofilm formation. The expression of both operons was severely reduced both in the laboratory and a non-domesticated strain upon accumulation of c-di-AMP. In excellent agreement, the corresponding strain was unable to form complex colonies. In B. subtilis, the transcription factor SinR controls the expression of biofilm genes by binding to their promoter regions resulting in transcription repression. Inactivation of the sinR gene restored biofilm formation even at high intracellular c-di-AMP concentrations suggesting that the second messenger acts upstream of SinR in the signal transduction pathway. As c-di-AMP accumulation did not affect the intracellular levels of SinR, we conclude that the nucleotide affects the activity of SinR. PMID:27252699

  20. cAMP Modulates Macrophage Development by Suppressing M-CSF-Induced MAPKs Activation

    Institute of Scientific and Technical Information of China (English)

    Ning Zhu; Jian Cui; Chunxia Qiao; Yan Li; Yuanfang Ma; Jiyan Zhang; Beifen Shen

    2008-01-01

    M-CSF is a key cytokine in macrophage development by inducing MAPKs activation, and cAMP can inhibit MAPKs activation induced by inflammatory stimuli. To explore the effects of cAMP on M-CSF-induced MAPKs activation and on macrophage development, the model of bone marrow-derived murine macrophages (BMMs) was used. The effects of cAMP on M-CSF-induced MAPKs activation were analyzed by Western blotting assay, and the effects of cAMP on CD14 and F4/80 expression during macrophage development were examined by FACS analysis.Macrophage morphology showed the successful establishment of the model of macrophage development. Western blotting assay revealed that M-CSF activated ERK, JNK and p38 in both mature and immature macrophages, and cAMP inhibited M-CSF-induced ERK, JNK and p38 activation in a time-dependent manner. FACS analysis revealed that macrophage development was impaired with cAMP pretreatment. In conclusion, cAMP modulates macrophage development by suppressing M-CSF-induced MAPKs activation.

  1. Hypoxia inhibits colonic ion transport via activation of AMP kinase.

    LENUS (Irish Health Repository)

    Collins, Danielle

    2012-02-01

    BACKGROUND AND AIMS: Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. METHODS: The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. RESULTS: This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. CONCLUSION: We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

  2. Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    M Shanthi

    2012-01-01

    Full Text Available Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR. Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25 and K. pneumoniae (n = 52 were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI guidelines. Minimum inhibitory concentration (MIC to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL. Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test. Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii, DHA (Dhahran Hospital, Saudi Arabia, ACC (Ambler class C, EBC (Amp C origin - Enterobacter cloacae groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

  3. Study on the synthesis of AMP derivatives for labeling with 153Sm and 166Ho

    International Nuclear Information System (INIS)

    This study describes the synthesis method and characterization of AMP-( Aminomethylene phosphonic acid): (PDTMP; BDTMP; DMPDTMP). AMP is synthesized by the condensation of correlative diamine, phosphorous acid and formaldehyde using a modified Mannich reaction in the presence of hydrochloric acid. Recrystallization of the crude product from water yields white crystals of pure legend, and subsequently characterized using 1H-NMR, IR spectroscopy, melting point, crystal picture, element analysis, metal trace analysis. Synthesized AMP, when tagged with therapeutic radio nuclides such as 153Sm and 166Ho are quite good. Complexes with RC purity and labeling efficiency 20 - 98% and above could be prepared by ordinary reaction condition. (author)

  4. Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition

    OpenAIRE

    Molina, Raul; Han, Dong-Yun; Su, Xue-Feng; Zhao, Run-Zhen; Zhao, Meimi; Sharp, Gretta M.; Chang, Yongchang; Ji, Hong-Long

    2011-01-01

    External Na+ self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αβγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβγ ENaC in a dose- a...

  5. cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids

    DEFF Research Database (Denmark)

    Madsen, Lise; Pedersen, Lone Møller; Liaset, Bjørn;

    2008-01-01

    The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti...... pivotal in regulating the adipogenic effect of n-6 PUFAs and that diet-induced differences in cAMP levels may explain the ability of n-6 PUFAs to either enhance or counteract adipogenesis and obesity....

  6. Effect of cAMP on short-circuit current in isolated human ciliary body

    Institute of Scientific and Technical Information of China (English)

    WU Ren-yi; MA Ning; HU Qian-qian

    2013-01-01

    Background Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current,Isc) across the ciliary processes in pigs.The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.Methods In an Ussing-type chamber system,the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed.The involvement of Cl-component in the bath solution was investigated.The effect of Cl-channel (10 μmol/L niflumic acid and 1 mmol/L 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)),K+ channel (10 mmol/L tetraethylammonium chloride (TEA)),or Na+ channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.Results Dose-dependently,8-bromo-cAMP (10 nmol/L-30 μmol/L) or forskolin (10 nmol/L-3 μmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue.Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side.When the tissue was bathed in low Cl-solutions,the Isc increase was significantly inhibited.Finally,niflumic acid and DIDS,but not TEA or amiloride,significantly prevented the Isc increase induced by 8-bromo-cAMP.Conclusions cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes.Chloride is likely to be among the ions,the transportation of which across the tissue is triggered by cAMP,suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

  7. New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

    Science.gov (United States)

    Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

    2016-08-01

    Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

  8. Cyclic AMP-dependent protein kinase I: Cyclic nucleotide binding, structural changes, and release of the catalytic subunits

    OpenAIRE

    Smith, Stephen B.; White, Hillary D.; Siegel, Jeffrey B.; Krebs, Edwin G.

    1981-01-01

    Type I cyclic AMP (cAMP)-dependent protein kinase is composed of a dimeric regulatory subunit (R2) and two catalytic subunits (C subunits). The R2 dimer binds four cAMP molecules to release the two C subunits. To characterize the cAMP binding sites and elucidate their role in the release of the C subunits, the R2 dimer has been studied by equilibrium methods. The cAMP titration of R2 was monitored by endogenous tryptophan fluorescence, and the results suggest one class of binding sites. The t...

  9. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  10. Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)

    Science.gov (United States)

    Kaye, Karl; Bryant, David E.; Marriott, Katie E. R.; Ohara, Shohei; Fishwick, Colin W. G.; Kee, Terence P.

    2016-05-01

    We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H2P2O5 2-; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M2+) and by organic co-factors such as acetate (AcO-). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M2+ & AcO-. Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

  11. 5 '-AMP impacts lymphocyte recirculation through activation of A(2B) receptors

    NARCIS (Netherlands)

    Bouma, Hjalmar R.; Mandl, Judith N.; Strijkstra, Arjen M.; Boerema, Ate S.; Kok, Jan-Willem; van Dam, Annie; IJzerman, Ad; Kroese, Frans G. M.; Henning, Robert H.

    2013-01-01

    Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5-AMP. Pr

  12. Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

    Science.gov (United States)

    Pinkney, M; Hoggett, J G

    1988-03-15

    Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase. PMID:2839152

  13. The effects of DP and AMP on immune functions in irradiated mice

    International Nuclear Information System (INIS)

    The effects of DP and AMP on the immune function in X-ray irradiated mice with different doses were studied in this dissertation. Mice (BALB/C) were irradiated with different doses (0, 1, 2, 4 Gy) of X-rays. The spontaneous proliferation of thymocytes and ConA-induced proliferation reaction of splenocytes in mice pretreated with DP( 2 mg) and AMP (5 mg) were detected. The results were as follows: the spontaneous proliferation of the thymocytes and the ConA-induced proliferation reaction of splenocytes in mice injected with DP and AMP decreased markedly, but increased significantly in mice treated with DP and AMP prior to irradiation by 1Gy and 2Gy X-rays

  14. 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

  15. The role of AMP-activated protein kinase in regulation of skeletal muscle metabolism

    OpenAIRE

    Anna Dziewulska; Paweł Dobrzyń; Agnieszka Dobrzyń

    2010-01-01

    AMP-activated protein kinase (AMPK) is a conserved, ubiquitously expressed eukaryotic enzyme that is activated in response to increasing AMP level. Regulation of AMPK activity in skeletal muscle is coordinated by contraction and phosphorylation by upstream kinases and a growing number of hormones and cytokines. Once activated, AMPK turns on catabolic, ATP-generating pathways, and turns off ATP-consuming metabolic processes such as biosynthesis and proliferation. Activation of AMPK promotes gl...

  16. Cyclic AMP concentrations in dendritic cells induce and regulate Th2 immunity and allergic asthma

    OpenAIRE

    Lee, Jihyung; Kim, Tae Hoon; Murray, Fiona; Li, Xiangli; Choi, Sara S.; Broide, David H.; Corr, Maripat; Lee, Jongdae; Webster, Nicholas J. G.; Insel, Paul A.; Raz, Eyal

    2015-01-01

    Allergic asthma is characterized by Th2 type inflammation, leading to airway hyperresponsiveness and remodeling. However, the mechanisms by which DC promote Th2 differentiation remain unclear. Herein we demonstrate that low cAMP levels in DC induce Th2-biased responses in vitro and in vivo. Furthermore, mice with conditional deletion of Gnas in DC (GnasΔCD11c mice) develop spontaneous bronchial asthma that shares multiple similarities with human asthma. In contrast, increasing cAMP levels inh...

  17. Blockade of beta-adrenoceptors enhances cAMP signal transduction in vivo

    Science.gov (United States)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1998-01-01

    The aim of this study was to determine whether the blockade of beta-adrenoceptors would enhance cAMP-mediated signal transduction processes in vivo. The administration of the membrane permeable cAMP analogue, 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP, 10 micromol/kg, i.v.) produced an increase in heart rate (+27 +/- 2%, P < 0.05), a fall in mean arterial blood pressure (-21 +/- 3%, P < 0.05) and falls in hindquarter (-12 +/- 3%, P < 0.05) and mesenteric (-32 +/- 3%, P < 0.05) vascular resistances in pentobarbital-anesthetized rats. The beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.) lowered heart rate (-12 +/- 3%, P < 0.05) but did not affect mean arterial blood pressure or vascular resistances. The tachycardia, hypotension and vasodilation produced by 8-CPT-cAMP were exaggerated after administration of propranolol (P < 0.05 for all comparisons). The nitric oxide-donor, sodium nitroprusside (2 microg/kg, i.v.), produced falls in mean arterial blood pressure and vascular resistances of similar magnitude to those produced by 8-CPT-cAMP. These sodium nitroprusside-induced responses were unaffected by propranolol (P < 0.05 for all comparisons). Sodium nitroprusside also produced a minor increase in heart rate (+5 +/- 1%, P < 0.05) which was abolished by propranolol. These findings suggest that 8-CPT-cAMP directly increases heart rate and that blockade of beta-adrenoceptors enhances the potency of cAMP within the heart and vasculature.

  18. 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.

  19. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs).

    Science.gov (United States)

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina

    2014-10-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  20. Pharmacological elevation of cyclic AMP and transmitter release at the mouse neuromuscular junction.

    Science.gov (United States)

    Dryden, W F; Singh, Y N; Gordon, T; Lazarenko, G

    1988-03-01

    Intracellular recordings of spontaneous and evoked end-plate potentials have been made at the neuromuscular junction of mouse hemidiaphragms to determine a possible role of cyclic AMP (cAMP) in the release of acetylcholine from presynaptic terminals. Spontaneous release, as determined from the frequency of miniature end-plate potentials, was increased by drugs that inhibit phosphodiesterase: isobutylmethylxanthine (IBMX), SQ 20,009, theophylline, and caffeine; drugs that stimulate adenylate cyclase: forskolin, fluoride, and cholera toxin, and the stable analogue of cAMP: 8-bromo-cAMP but not dibutyryl cAMP. Release increased with time during maintained exposure to the drugs and generally followed a simple exponential time course with time constants ranging from 8 to 17 min at 20 degrees C, except for SQ 20,009 and cholera toxin which required longer exposure times for effect. The order of potency of the phosphodiesterase inhibitors was IBMX = SQ 20,009 greater than theophylline = caffeine. This is consistent with an effect mediated by an increase in cAMP concentrations within the nerve terminal. Evoked release, determined from the quantal content of the end-plate potential, was increased to a lesser extent than spontaneous release. The results are discussed with reference to the possible involvement of second messengers in the release of vesicles from nerve terminals in vertebrate synapses.

  1. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.

    Directory of Open Access Journals (Sweden)

    Davide Calebiro

    2009-08-01

    Full Text Available G-protein-coupled receptors (GPCRs are generally thought to signal to second messengers like cyclic AMP (cAMP from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein alpha(s-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell.

  2. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Science.gov (United States)

    Aykaç Fas, Burcu; Tutar, Yusuf; Haliloğlu, Türkan

    2013-01-01

    Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  3. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, L; Martinussen, J; Møllegaard, N E;

    1990-01-01

    AMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets...

  4. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Science.gov (United States)

    Bacallao, Ketty; Monje, Paula V

    2015-01-01

    Isolated Schwann cells (SCs) respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1). To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP) and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC) agonists and antagonists revealed that selective transmembrane AC (tmAC) activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC), a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the uncoupling of signals

  5. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

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    Ketty Bacallao

    Full Text Available Isolated Schwann cells (SCs respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1. To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC agonists and antagonists revealed that selective transmembrane AC (tmAC activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC, a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the

  6. Inhibition of Vascular Smooth Muscle Growth via Signaling Crosstalk between AMP-Activated Protein Kinase and cAMP-Dependent Protein Kinase

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    Joshua Daniel Stone

    2012-10-01

    Full Text Available Abnormal vascular smooth muscle (VSM growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK and cAMP-dependent protein kinase (PKA. Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remains unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells, the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSM cell migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashions. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.

  7. Nuclease-Resistant c-di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors.

    Science.gov (United States)

    Meehan, Robert E; Torgerson, Chad D; Gaffney, Barbara L; Jones, Roger A; Strobel, Scott A

    2016-02-16

    The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3'-5'-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activates specific pathways and mediates phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogues, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position more than a carbonyl at the C6 position. We also identified phosphate-modified analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues exhibited a substantial decrease in ydaO affinity but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways. PMID:26789423

  8. Fecal Colonization with Extended-Spectrum Beta-Lactamase and AmpC-Producing Escherichia coli

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    Mohamed H. Al-Agamy

    2016-01-01

    Full Text Available Background. Extended-spectrum β-lactamases (ESβLs and AmpC β-lactamases cause β-lactam resistance in Escherichia coli. Fecal colonization by ESβL- and/or AmpC-positive E. coli is a source of nosocomial infections. Methods. In order to investigate inpatient fecal colonization by ESβLs and AmpC, antibiotic sensitivity tests were conducted and minimum inhibitory concentrations (MICs were determined using the disk diffusion method and E-test, respectively. Characterization of ESβL and AmpC was performed using E-test strips, and a set of PCRs and DNA sequence analyses were used to characterize the ESβL and AmpC genes. Results. The whole collection of E. coli isolates (n=50 was sensitive to imipenem, tigecycline, colistin, and fosfomycin, while 26% of the isolates showed reduced susceptibility to ceftazidime (MIC ≥ 4 μg/mL. ESβL was phenotypically identified in 26% (13/50 of cases, while AmpC activity was detected in two ESβL-producing E. coli isolates. All ESβL-producing E. coli were positive for the CTX-M gene, eleven isolates carried blaCTX-M-15, and two isolates carried blaCTX-M-14 gene. Two CTX-M-positive E. coli isolates carried blaCMY-2. Conclusions. The alimentary tract is a significant reservoir for ESβL- and/or AmpC-producing E. coli, which may lead to nosocomial infection.

  9. Fecal Colonization with Extended-Spectrum Beta-Lactamase and AmpC-Producing Escherichia coli.

    Science.gov (United States)

    Al-Agamy, Mohamed H; El Mahdy, Taghrid S; Shibl, Atef M

    2016-01-01

    Background. Extended-spectrum β-lactamases (ESβLs) and AmpC β-lactamases cause β-lactam resistance in Escherichia coli. Fecal colonization by ESβL- and/or AmpC-positive E. coli is a source of nosocomial infections. Methods. In order to investigate inpatient fecal colonization by ESβLs and AmpC, antibiotic sensitivity tests were conducted and minimum inhibitory concentrations (MICs) were determined using the disk diffusion method and E-test, respectively. Characterization of ESβL and AmpC was performed using E-test strips, and a set of PCRs and DNA sequence analyses were used to characterize the ESβL and AmpC genes. Results. The whole collection of E. coli isolates (n = 50) was sensitive to imipenem, tigecycline, colistin, and fosfomycin, while 26% of the isolates showed reduced susceptibility to ceftazidime (MIC ≥ 4 μg/mL). ESβL was phenotypically identified in 26% (13/50) of cases, while AmpC activity was detected in two ESβL-producing E. coli isolates. All ESβL-producing E. coli were positive for the CTX-M gene, eleven isolates carried bla CTX-M-15, and two isolates carried bla CTX-M-14 gene. Two CTX-M-positive E. coli isolates carried bla CMY-2. Conclusions. The alimentary tract is a significant reservoir for ESβL- and/or AmpC-producing E. coli, which may lead to nosocomial infection. PMID:27340657

  10. Biochemical characterization and cellular imaging of a novel, membrane permeable fluorescent cAMP analog

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    Zaccolo Manuela

    2008-06-01

    Full Text Available Abstract Background A novel fluorescent cAMP analog (8-[Pharos-575]- adenosine-3', 5'-cyclic monophosphate was characterized with respect to its spectral properties, its ability to bind to and activate three main isoenzymes of the cAMP-dependent protein kinase (PKA-Iα, PKA-IIα, PKA-IIβ in vitro, its stability towards phosphodiesterase and its ability to permeate into cultured eukaryotic cells using resonance energy transfer based indicators, and conventional fluorescence imaging. Results The Pharos fluorophore is characterized by a Stokes shift of 42 nm with an absorption maximum at 575 nm and the emission peaking at 617 nm. The quantum yield is 30%. Incubation of the compound to RIIα and RIIβ subunits increases the amplitude of excitation and absorption maxima significantly; no major change was observed with RIα. In vitro binding of the compound to RIα subunit and activation of the PKA-Iα holoenzyme was essentially equivalent to cAMP; RII subunits bound the fluorescent analog up to ten times less efficiently, resulting in about two times reduced apparent activation constants of the holoenzymes compared to cAMP. The cellular uptake of the fluorescent analog was investigated by cAMP indicators. It was estimated that about 7 μM of the fluorescent cAMP analog is available to the indicator after one hour of incubation and that about 600 μM of the compound had to be added to intact cells to half-maximally dissociate a PKA type IIα sensor. Conclusion The novel analog combines good membrane permeability- comparable to 8-Br-cAMP – with superior spectral properties of a modern, red-shifted fluorophore. GFP-tagged regulatory subunits of PKA and the analog co-localized. Furthermore, it is a potent, PDE-resistant activator of PKA-I and -II, suitable for in vitro applications and spatial distribution evaluations in living cells.

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

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    Andre Terzic

    2009-04-01

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

  12. The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas.

    Science.gov (United States)

    Du, Qingyou; Schilde, Christina; Birgersson, Elin; Chen, Zhi-hui; McElroy, Stuart; Schaap, Pauline

    2014-02-01

    Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.

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

    KAUST Repository

    Thomas, Ludivine

    2013-05-01

    The second messenger 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses in higher plants. Here we used proteomics to identify cAMP-dependent protein signatures in Arabidopsis thaliana and identify a number of differentially expressed proteins with a role in light- and temperature-dependent responses, notably photosystem II subunit P-1, plasma membrane associated cation-binding protein and chaperonin 60 β. Based on these proteomics results we conclude that, much like in cyanobacteria, algae and fungi, cAMP may have a role in light signaling and the regulation of photosynthesis as well as responses to temperature and we speculate that ACs could act as light and/or temperature sensors in higher plants. Biological significance: This current study is significant since it presents the first proteomic response to cAMP, a novel and key second messenger in plants. It will be relevant to researchers in plant physiology and in particular those with an interest in second messengers and their role in biotic and abiotic stress responses. © 2013 Elsevier B.V.

  14. Strain-dependent occurrence of functional GTP:AMP phosphotransferase (AK3) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Schricker, R; Magdolen, V; Strobel, G; Bogengruber, E; Breitenbach, M; Bandlow, W

    1995-12-29

    The gene for yeast GTP:AMP phosphotransferase (PAK3) was found to encode a nonfunctional protein in 10 laboratory strains and one brewers' strain. The protein product showed high similarity to vertebrate AK3 and was located exclusively in the mitochondrial matrix. The deduced amino acid sequence revealed a protein that was shorter at the carboxyl terminus than all other known adenylate kinases. Introduction of a +1 frameshift into the 3'-terminal region of the gene extended homology of the deduced amino acid sequence to other members of the adenylate kinase family including vertebrate AK3. Frameshift mutations obtained after in vitro and in vivo mutagenesis were capable of complementing the adk1 temperature-conditional deficiency in Escherichia coli, indicating that the frameshift led to the expression of a protein that could phosphorylate AMP. Some yeasts, however, including strain D273-10B, two wine yeasts, and two more distantly related yeast genera, harbored an active allele, named AKY3, which contained a +1 frameshift close to the carboxyl terminus as compared with the laboratory strains. The encoded protein exhibited GTP:AMP and ITP:AMP phosphotransferase activities but did not accept ATP as phosphate donor. Although single copy in the haploid genome, disruption of the AKY3 allele displayed no phenotype, excluding the possibility that laboratory and brewers' strains had collected second site suppressors. It must be concluded that yeast mitochondria can completely dispense with GTP:AMP phosphotransferase activity.

  15. Effects of radioprotectors on the cAMP and cGMP systems

    International Nuclear Information System (INIS)

    The sulphur-containing radioprotectors mercaptoethylamine (MEA), aminoethylisothiourea (AET), 2-aminothiazoline, 4-oxo-2-amino-thiazoline, and S-S-oxapentane-1,5-diisorthiourea, and the radioprotective biogenic amines serotonin, histamine, and dopamine, caused the elevation of cAMP content and intensified the rate of cAMP-dependent protein phosphorylation in tissues of animals following intraperitoneal injection at radioprotective doses. Biogenic amines stimulated the adenylate cyclase activity in membrane preparations from liver, spleen, and small-intestine mucosa; sulphur-containing radioprotectors caused no such effects. None of the radioprotectors affected CAMP and cGMP phosphodiesterases in vitro. AET and MEA inhibited guanylate cyclase in vitro, whereas serotonin and dopamine stimulated the enzyme. A biphasic change in the level of cGMP was observed in tissues after the administration of MEA and AET (more than 2-fold fally by 1-3 min after the administration of drug and 1.4-fold rise after 15-20 min); serotonin and dopamine caused a slow rise in the cGMP level, the cAMP/cGMP ratio in liver showed biphasic changes in level during the 20 min following injection of serotonin. The data obtained support the conclusion that the action of radioprotectors on cellular metabolism in animals may be mediated by the cAMP system.The reciprocal regulation of radioresistance by cAMP and cGMP is unlikely to exist. (orig.)

  16. Diatom acclimation to elevated CO2 via cAMP signalling and coordinated gene expression

    Science.gov (United States)

    Hennon, Gwenn M. M.; Ashworth, Justin; Groussman, Ryan D.; Berthiaume, Chris; Morales, Rhonda L.; Baliga, Nitin S.; Orellana, Mónica V.; Armbrust, E. V.

    2015-08-01

    Diatoms are responsible for ~40% of marine primary productivity, fuelling the oceanic carbon cycle and contributing to natural carbon sequestration in the deep ocean. Diatoms rely on energetically expensive carbon concentrating mechanisms (CCMs) to fix carbon efficiently at modern levels of CO2 (refs , , ). How diatoms may respond over the short and long term to rising atmospheric CO2 remains an open question. Here we use nitrate-limited chemostats to show that the model diatom Thalassiosira pseudonana rapidly responds to increasing CO2 by differentially expressing gene clusters that regulate transcription and chromosome folding, and subsequently reduces transcription of photosynthesis and respiration gene clusters under steady-state elevated CO2. These results suggest that exposure to elevated CO2 first causes a shift in regulation, and then a metabolic rearrangement. Genes in one CO2-responsive cluster included CCM and photorespiration genes that share a putative cAMP-responsive cis-regulatory sequence, implying these genes are co-regulated in response to CO2, with cAMP as an intermediate messenger. We verified cAMP-induced downregulation of CCM gene δ-CA3 in nutrient-replete diatom cultures by inhibiting the hydrolysis of cAMP. These results indicate an important role for cAMP in downregulating CCM and photorespiration genes under elevated CO2 and provide insights into mechanisms of diatom acclimation in response to climate change.

  17. Intercellular redistribution of cAMP underlies selective suppression of cancer cell growth by connexin26.

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    Anjana Chandrasekhar

    Full Text Available Connexins (Cx, which constitute gap junction intercellular channels in vertebrates, have been shown to suppress transformed cell growth and tumorigenesis, but the mechanism(s still remain largely speculative. Here, we define the molecular basis by which Cx26, but less frequently Cx43 or Cx32, selectively confer growth suppression on cancer cells. Functional intercellular coupling is shown to be required, producing partial blocks of the cell cycle due to prolonged activation of several mitogenic kinases. PKA is both necessary and sufficient for the Cx26 induced growth inhibition in low serum and the absence of anchorage. Activation of PKA was not associated with elevated cAMP levels, but appeared to result from a redistribution of cAMP throughout the cell population, eliminating the cell cycle oscillations in cAMP required for efficient cell cycle progression. Cx43 and Cx32 fail to mediate this redistribution as, unlike Cx26, these channels are closed during the G2/M phase of the cell cycle when cAMP levels peak. Comparisons of tumor cell lines indicate that this is a general pattern, with growth suppression by connexins occurring whenever cAMP oscillates with the cell cycle, and the gap junction remain open throughout the cell cycle. Thus, gap junctional coupling, in the absence of any external signals, provides a general means to limit the mitotic rate of cell populations.

  18. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

    International Nuclear Information System (INIS)

    Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases

  19. cAMP signalling in mushroom bodies modulates temperature preference behaviour in Drosophila.

    Science.gov (United States)

    Hong, Sung-Tae; Bang, Sunhoe; Hyun, Seogang; Kang, Jongkyun; Jeong, Kyunghwa; Paik, Donggi; Chung, Jongkyeong; Kim, Jaeseob

    2008-08-01

    Homoiotherms, for example mammals, regulate their body temperature with physiological responses such as a change of metabolic rate and sweating. In contrast, the body temperature of poikilotherms, for example Drosophila, is the result of heat exchange with the surrounding environment as a result of the large ratio of surface area to volume of their bodies. Accordingly, these animals must instinctively move to places with an environmental temperature as close as possible to their genetically determined desired temperature. The temperature that Drosophila instinctively prefers has a function equivalent to the 'set point' temperature in mammals. Although various temperature-gated TRP channels have been discovered, molecular and cellular components in Drosophila brain responsible for determining the desired temperature remain unknown. We identified these components by performing a large-scale genetic screen of temperature preference behaviour (TPB) in Drosophila. In parallel, we mapped areas of the Drosophila brain controlling TPB by targeted inactivation of neurons with tetanus toxin and a potassium channel (Kir2.1) driven with various brain-specific GAL4s. Here we show that mushroom bodies (MBs) and the cyclic AMP-cAMP-dependent protein kinase A (cAMP-PKA) pathway are essential for controlling TPB. Furthermore, targeted expression of cAMP-PKA pathway components in only the MB was sufficient to rescue abnormal TPB of the corresponding mutants. Preferred temperatures were affected by the level of cAMP and PKA activity in the MBs in various PKA pathway mutants. PMID:18594510

  20. Improved molecular toolkit for cAMP studies in live cells

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    Nicol Xavier

    2011-07-01

    Full Text Available Abstract Background cAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations. Findings We have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments. Conclusions The modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.

  1. cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

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    Ghania Ramdani

    2015-05-01

    Full Text Available Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

  2. Actin induction during PMA and cAMP-dependent signal pathway activation in Entamoeba histolytica trophozoites.

    Science.gov (United States)

    Ortiz, D; del Carmen Dominguez-Robles, M; Villegas-Sepúlveda, N; Meza, I

    2000-10-01

    Activation of PKC or cAMP-dependent signalling pathways in Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rearrangements necessary for adhesion and locomotion. Analogous motifs to SRE and CRE sequences--known to respond to PMA and cAMP--were identified within the 5' regulatory region (5'RR) of one of the parasite actin genes. These sequences could be involved in the actin transcriptional upregulation reported during signalling. To test this hypothesis, a plasmid containing the 5'RR of the actin gene fused to the bacterial neomycin gene (neo) was used for stable transfection. Expression of neo and endogenous actin was measured after stimulation of transfected amoebae by PMA and dcAMP. It was found that both compounds induced neo and actin expression and showed a co-operative effect in the induction of neo. Induction by PMA or dcAMP failed if the directing amoebic 5'RR lacked SRE and CRE motifs. Transfection of amoebae with plasmid constructs, containing either progressive deletions of the actin 5'RR or site-directed mutations of the SRE and CRE-like motifs, corroborated that these sequences and a co-ordinated participation of PKC- and PKA-activated transcription factors are responsible for the increments in neo and actin mRNAs. In vivo, these PMA and cAMP-response elements could play an important role in regulating actin expression and organization in signalling processes activated during tissue invasion.

  3. AMP makes native snake muscle fructose-1,6-bisphosphatase to an alkaline enzyme

    Institute of Scientific and Technical Information of China (English)

    赵辅昆; 徐松琴; 杜立林; 许根俊

    2000-01-01

    A substance in the crude preparation of NADP+ has been found, which activates snake muscle fructose-1,6-bisphosphatase at pH 9.2 and inhibits the enzyme at pH 7.5. After isolation and extensive characterization, the substance has been determined to be AMP. The activation depends on the concentrations of Mg2+ and could be observed only at concentrations above 1 mmol/L. In the presence of AMP, snake muscle fructose-1,6-bisphosphatase resembles an alkaline enzyme. Kinetic studies indicate that AMP and Mg2+ competitively regulate the activity of the enzyme. AMP releases the inhibition of Mg2+ at high concentration at alkaline pH. It has been reported that fructose-1,6-bisphosphatase with a pH optimum in the alkaline region is caused by limited proteolysis. AMP is also able to make fructose-1,6-bisphosphatase to be an alkaline enzyme. This finding indicates that proteolysis may not be the only reason for shift of the optimum pH of fructose-1,6-bisphosphatase to alkaline side and it may imply some significanc

  4. Characterization of chromosomal qnrB and ampC alleles in Citrobacter freundii isolates from different origins.

    Science.gov (United States)

    Liao, Xiaoping; Fang, Liangxing; Li, Liang; Sun, Jian; Li, Xingping; Chen, Muya; Deng, Hui; Yang, Qiu'e; Li, Xue; Liu, Yahong

    2015-10-01

    The association of ESBLs (extended-spectrum beta-lactamases)/pAmpCs (plasmid-mediated AmpC β-lactamases) with PMQR (plasmid mediated quinolone resistance) in gram-negative bacteria has been of great concern. The present study was performed to characterize the diversity, gene location, genetic context, and evolution of ampC and qnrB alleles in isolates of Citrobacter freundii. Fifteen isolates of C. freundii were identified from a total of 788 isolates of Enterobacteriaceae derived from humans, animals, animal food products, and the environment between 2010 and 2012. Co-existence of qnrB/ΔqnrB with ampC was detected in all C. freundii isolates. Both ampC and qnrB genes were found to be located on the chromosome, but were distantly separated on the chromosome. Seven and six novel alleles were discovered for the 10 ampC and qnrB variants detected in this study, respectively. Phylogenetic analysis showed that the new alleles differed a little from the variants of ampC/qnrB previously described in this genus. The genetic context surrounding ampC genes was AmpR-AmpC-Blc-SugE. However, five different genetic contexts surrounding qnrB/ΔqnrB genes were observed, but they occurred in all cases between the pspF and sapA genes. Additionally, cloning experiments showed that the regions containing different qnrB alleles, even with different genetic contexts, contributed to the reduction of quinolone susceptibility. Our results showed that the chromosomal ampC and qnrB alleles are closely related to C. freundii. However, unlike ampC, qnrB alleles seemed to be related to the genetic contexts surrounding them. The evolution of these two genes in C. freundii isolates might be through different pathways.

  5. AMP-activated protein kinase phosphorylation in brain is dependent on method of sacrifice and tissue preparation

    OpenAIRE

    Scharf, Matthew T.; Mackiewicz, Miroslaw; Naidoo, Nirinjini; O'Callaghan, James P.; Pack, Allan I.

    2007-01-01

    AMP-activated protein kinase is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α-AMP-activated protein kinase phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α-AMP-activated protein kinase phosphorylation in the mouse brain, we compared different methods of sacrifice and tissue preparation. We found that fre...

  6. High prevalence of fecal carriage of extended spectrum β-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs

    OpenAIRE

    JoostHordijk; CindyDierikx

    2013-01-01

    Extended-spectrum-β-lactamase (ESBL)/AmpC producing Enterobacteriaceae have been reported worldwide amongst isolates obtained from humans, food-producing animals, companion animals, and environmental sources. However, data on prevalence of fecal carriage of ESBL/AmpC producing Enterobacteriaceae in healthy companion animals is limited. This pilot study describes the prevalence of ESBL/AmpC encoding genes in healthy cats and dogs, and cats and dogs with diarrhea. Twenty fecal samples of each g...

  7. High prevalence of fecal carriage of extended spectrum beta-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs

    OpenAIRE

    Hordijk, J.; Schoormans, A.; Kwakernaak, M.; Duim, B.; Broens, E.; Dierikx, C.M.; Mevius, D.J.; Wagenaar, J. A.

    2013-01-01

    Extended-spectrum-ß-lactamase (ESBL)/AmpC producing Enterobacteriaceae have been reported worldwide amongst isolates obtained from humans, food-producing animals, companion animals, and environmental sources. However, data on prevalence of fecal carriage of ESBL/AmpC producing Enterobacteriaceae in healthy companion animals is limited. This pilot study describes the prevalence of ESBL/AmpC encoding genes in healthy cats and dogs, and cats and dogs with diarrhea. Twenty fecal samples of each g...

  8. Effect of cholera toxin on cAMP levels and Na/sup +/ influx in isolated intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hyun, C.S.; Kimmich, G.A.

    1982-09-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 ..mu..g/ml cholera toxin (CT) at 37/sup 0/C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na/sup +/ influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na/sup +/ entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na/sup +/ during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na/sup +/ influx suggest that the reactivation of the Na/sup +/ transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP.

  9. The importance of dietary modulation of cAMP and insulin signaling in adipose tissue and the development of obesity

    DEFF Research Database (Denmark)

    Madsen, Lise; Kristiansen, Karsten

    2010-01-01

    Adipose tissue plays a pivotal role in whole body energy homeostasis. In this review, we summarize knowledge of the seemingly paradoxical roles of insulin and cyclic adenosine monophosphate (cAMP) signaling in adipocyte differentiation and function, emphasizing the interplay between the two...... branches of cAMP signaling, the canonical protein kinase A-dependent pathways and the novel exchange protein activated by cAMP (Epac)-dependent pathways, and insulin signaling. We discuss how macronutrients via changes in the balance between insulin- and cAMP-dependent signaling can affect the development...

  10. A Single Stage Low Gain Pseudo Differential Class- AB telescopic Cascoded Op-amp for Pipelined ADC

    Directory of Open Access Journals (Sweden)

    D.S.Shylu Dr.D.Jackuline Moni 2 Anita Antony 3 A J Sowjanya.K 4 Neetha C John 5

    2012-12-01

    Full Text Available In this paper, a low gain pseudo differential classAB telescopic op-amp for pipelined ADC is presented. By applying split capacitor CDS technique in a pipelined ADC, finite op-amp gain errors of low gain op-amps are eliminated. This significantly reduces the power consumption of the pipelined ADC. A pseudo differential class AB opamp is chosen as the suitable op-amp topology because of its high power efficiency and large signal swing compared to other topologies.Additional class AB capacitors used further enhances the DC gain and bandwidth of the amplifier.

  11. Emergence of Escherichia coli Producing Extended-Spectrum AmpC beta-lactamases (ESAC) in animals

    OpenAIRE

    Jean-YvesMADEC

    2014-01-01

    In both humans and animals, the spread of Extended-Spectrum β-Lactamases (ESBL)/AmpC producers has become a major issue, particularly due to the plasmidic dissemination of most of these genes. Besides, over-expression of the chromosomal ampC gene was largely reported in human and animal Enterobacteriaceae and, more recently, modifications within the coding region of the ampC gene [encoding Extended-spectrum AmpC β-lactamases (ESACs)] were shown to be responsible for an hydrolysis spectrum exp...

  12. Cystic Fibrosis Gene Encodes a cAMP-Dependent Chloride Channel in Heart

    Science.gov (United States)

    Hart, Padraig; Warth, John D.; Levesque, Paul C.; Collier, Mei Lin; Geary, Yvonne; Horowitz, Burton; Hume, Joseph R.

    1996-06-01

    cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significnatly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

  13. Involvement of the second messenger cAMP in gravity-signal transduction in physarum

    Science.gov (United States)

    Block, I.; Rabien, H.; Ivanova, K.

    The aim of the investigation was to clarify, whether cellular signal processing following graviperception involves second messenger pathways. The test object was a most gravisensitive free-living ameboid cell, the myxomycete (acellular slime mold) Physarum polycephalum. It was demonstrated that the motor response is related to acceleration-dependent changes in the levels of the cellular second messenger cyclic adenosine monophosphate (cAMP). Rotating Physarum plasmodia in the gravity field of the Earth about a horizontal axis increased their cAMP concentration. Depriving the cells for a few days of the acceleration stimulus (near weightlessness in a space experiment on STS-69) slightly lowered plasmodial cAMP levels. Thus, the results provide first indications that the acceleration-stimulus signal transduction chain of Physarum uses an ubiquitous second messenger pathway.

  14. Ultraviolet light induction of skin carcinoma in the mouse; influence of cAMP modifying agents.

    Science.gov (United States)

    Zajdela, F; Latarjet, R

    1978-01-01

    A short review of pathogenic factors in U.V. light skin carcinogenesis in the mouse is presented. Caffeine and theophylline applied locally during U.V. irradiation caused a 50 percent reduction of skin tumour induction in Swiss mice. These two chemicals are inhibitors of DNA postreplication repair, but they also raise the intracellular level of cyclic AMP by inhibiting cAMP phosphodiesterase with, as a consequence, a possible slowing down of cellular growth. Control experiments using three different chemicals capable of raising the cAMP level in epidermal cells gave negative results. These experimental data are compatible with our original hypothesis according to which production of skin cancers by U.V. radiation is in same way related to DNA repair which helps the cell to survive but allows or favours the occurrence of errors in cellular DNA.

  15. Autoregulation of PhoP/PhoQ and positive regulation of the cyclic AMP receptor protein-cyclic AMP complex by PhoP in Yersinia pestis.

    Science.gov (United States)

    Zhang, Yiquan; Wang, Li; Han, Yanping; Yan, Yanfeng; Tan, Yafang; Zhou, Lei; Cui, Yujun; Du, Zongmin; Wang, Xiaoyi; Bi, Yujing; Yang, Huiying; Song, Yajun; Zhang, Pingping; Zhou, Dongsheng; Yang, Ruifu

    2013-03-01

    Yersinia pestis is one of the most dangerous bacterial pathogens. PhoP and cyclic AMP receptor protein (CRP) are global regulators of Y. pestis, and they control two distinct regulons that contain multiple virulence-related genes. The PhoP regulator and its cognate sensor PhoQ constitute a two-component regulatory system. The regulatory activity of CRP is triggered only by binding to its cofactor cAMP, which is synthesized from ATP by adenylyl cyclase (encoded by cyaA). However, the association between the two regulatory systems PhoP/PhoQ and CRP-cAMP is still not understood for Y. pestis. In the present work, the four consecutive genes YPO1635, phoP, phoQ, and YPO1632 were found to constitute an operon, YPO1635-phoPQ-YPO1632, transcribed as a single primary RNA, whereas the last three genes comprised another operon, phoPQ-YPO1632, transcribed with two adjacent transcriptional starts. Through direct PhoP-target promoter association, the transcription of these two operons was stimulated and repressed by PhoP, respectively; thus, both positive autoregulation and negative autoregulation of PhoP/PhoQ were detected. In addition, PhoP acted as a direct transcriptional activator of crp and cyaA. The translational/transcriptional start sites, promoter -10 and -35 elements, PhoP sites, and PhoP box-like sequences were determined for these PhoP-dependent genes, providing a map of the PhoP-target promoter interaction. The CRP and PhoP regulons have evolved to merge into a single regulatory cascade in Y. pestis because of the direct regulatory association between PhoP/PhoQ and CRP-cAMP. PMID:23264579

  16. Alterations of cAMP-dependent signaling in dystrophic skeletal muscle

    Directory of Open Access Journals (Sweden)

    Rüdiger eRudolf

    2013-10-01

    Full Text Available Autonomic regulation processes in striated muscles are largely mediated by cAMP/PKA-signaling. In order to achieve specificity of signaling its spatial-temporal compartmentation plays a critical role. We discuss here how specificity of cAMP/PKA-signaling can be achieved in skeletal muscle by spatio-temporal compartmentation. While a microdomain containing PKA type I in the region of the neuromuscular junction is important for post-synaptic, activity-dependent stabilization of the nicotinic acetylcholine receptor, PKA type I and II microdomains in the sarcomeric part of skeletal muscle are likely to play different roles, including the regulation of muscle homeostasis. These microdomains are due to specific A-kinase anchoring proteins, like rapsyn and myospryn. Importantly, recent evidence indicates that compartmentation of the cAMP/PKA-dependent signaling pathway and pharmacological activation of cAMP production are aberrant in different skeletal muscles disorders. Thus, we discuss here their potential as targets for palliative treatment of certain forms of dystrophy and myasthenia. Under physiological conditions, the neuropeptide, α-calcitonin-related peptide, as well as beta-adrenergic agonists are the most-mentioned natural triggers for activating cAMP/PKA signaling in skeletal muscle. While the precise domains and functions of these first messengers are still under investigation, agonists of β2-adrenoceptors clearly exhibit anabolic activity under normal conditions and reduce protein degradation during atrophic periods. Past and recent studies suggest direct sympathetic innervation of skeletal muscle fibers. In summary, the organization and roles of cAMP-dependent signaling in skeletal muscle are increasingly understood, revealing crucial functions in processes like nerve-muscle interaction and muscle trophicity.

  17. The status of drug resistance and ampC gene expression in Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    周志慧; 李兰娟; 俞云松; 马亦林

    2003-01-01

    Objective To investigate the status of the drug resistance and the ampC gene expression of Enterobacter cloacae. Methods Disk diffusion tests were made for detecting the susceptibility of antimicrobial agents against Enterobacter cloacae. AmpC gene was amplified by polymerase chain reaction (PCR) and verified by DNA sequencing. AmpC gene expression was analyzed according to antimicrobial agent sensitive phenotype.Results The sensitivity rates of 144 strains to imipenam, cefepime and cefoperazone/sulbactam were 98.61%, 65.97% and 63.89%, respectively. The sensitivity rates of 144 strains to other antimicrobial agents were lower. Among the 144 strains 120 were found to be positive by PCR for ampC. The PCR product showed high homology to the GenBank ampC sequence. Stably derepressed strains, hyperinducible strains and unexpressing or lower level expressing strains accounted for 30.0% (36/120), 37.5% (45/120), and 32.5% (39/120), respectively. Fifty-six out of 120 strains (46.67%) also produced extended spectrum β-lactamases (ESBLs). The hyperinducible strains were highly sensitive to all the antimicrobial agents except amoxicillin/clavulanic acid and cefuroxime, while the stably derepressed strains were only sensitive to imipenam and cefepime. However, sensitivity to cefepime decreased if the strains also produced ESBLs. Conclusions The durg resistant status of Enterobacter cloacae is severe. Clearing out the expressive status of ampC gene will be helpful in selection of antimicrobial agents in the treatment of clinical infection.

  18. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Directory of Open Access Journals (Sweden)

    Burcu Aykaç Fas

    Full Text Available Escherichia coli cyclic AMP Receptor Protein (CRP undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD simulations and Gaussian Network Model (GNM. The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  19. Association of Novel Nonsynonymous Single Nucleotide Polymorphisms in ampD with Cephalosporin Resistance and Phylogenetic Variations in ampC, ampR, ompF, and ompC in Enterobacter cloacae Isolates That Are Highly Resistant to Carbapenems

    Science.gov (United States)

    Ellington, Matthew J.; Hopkins, Katie L.; Turton, Jane F.; Doumith, Michel; Loy, Richard; Staves, Peter; Hinic, Vladimira; Frei, Reno; Woodford, Neil

    2016-01-01

    In Enterobacter cloacae, the genetic lesions associated with derepression of the AmpC β-lactamase include diverse single nucleotide polymorphisms (SNPs) and/or indels in the ampD and ampR genes and SNPs in ampC, while diverse SNPs in the promoter region or SNPs/indels within the coding sequence of outer membrane proteins have been described to alter porin production leading to carbapenem resistance. We sought to define the underlying mechanisms conferring cephalosporin and carbapenem resistance in a collection of E. cloacae isolates with unusually high carbapenem resistance and no known carbapenemase and, in contrast to many previous studies, considered the SNPs we detected in relation to the multilocus sequence type (MLST)-based phylogeny of our collection. Whole-genome sequencing was applied on the most resistant isolates to seek novel carbapenemases, expression of ampC was measured by reverse transcriptase PCR, and porin translation was detected by SDS-PAGE. SNPs occurring in ampC, ampR, ompF, and ompC genes (and their promoter regions) were mostly phylogenetic variations, relating to the isolates' sequence types, whereas nonsynonymous SNPs in ampD were associated with derepression of AmpC and cephalosporin resistance. The additional loss of porins resulted in high-level carbapenem resistance, underlining the clinical importance of chromosomal mutations among carbapenem-resistant E. cloacae. PMID:26856839

  20. A Romantic life dedicated to Science: André-Marie Ampère's Autobiography

    OpenAIRE

    Martin Moruno, Dolorès

    2011-01-01

    This article explores André Marie Ampère's autobiography in order to analyse the dynamics of science in early 19th century French institutions. According to recent works that have emphasised the value of biographies in the history of science, this study examines Ampère's public self-representation to show the cultural transformations of a life dedicated to science in post-revolutionary French society. With this aim, I have interpreted this manuscript as an outstanding example of the scientific...

  1. Identification of a cAMP-dependent protein kinase in bovine and human follicular fluids.

    Science.gov (United States)

    Yang, L S; Kadam, A L; Koide, S S

    1993-11-01

    A soluble protein kinase (PK) was purified from bovine and human follicular fluids (FF) by ultrafiltration through a PM-10 membrane followed by chromatography on heparin-agarose, DEAE-cellulose and cellulose phosphate columns. The PK phosphorylated calf thymus histones and endogenous FF proteins having estimated Mrs of 40, 62, 128 and 180 KD. cAMP enhanced PK activity; whereas protein kinase A (PKA)-inhibitor peptide blocked the activity. The present findings suggest that the enzyme is a cAMP-dependent PK. PMID:8118427

  2. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

  3. Small-angle neutron scattering from poly(NIPA-co-AMPS) gels

    DEFF Research Database (Denmark)

    Travas-Sejdic, J.; Easteal, A.; Knott, R.;

    2000-01-01

    The microstructure of the poly( N-isopropylacrylamide-co-acrylamido- 2-methyl-1-propane sulphonic acid) gel, poly( NIPA-co-AMPS), was investigated as a function of temperature and cross-link density using the small angle neutron scattering technique. The sample temperature was varied in the range...... 30 to 55C. Two different behaviours of poly( NIPA-co-AMPS) gels were observed. At low temperature (30C), the magnitude of the scattered intensity increased with cross-link density suggesting that additional cross-links introduced more inhomogeneities in the gel network. At high temperatures the trend...

  4. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

    Directory of Open Access Journals (Sweden)

    Ketty Bacallao

    Full Text Available In Schwann cells (SCs, cyclic adenosine monophosphate (cAMP not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA and the exchange protein activated by cAMP (EPAC, on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical cAMP-specific pathway that is partially transduced by EPAC.

  5. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

    Science.gov (United States)

    Bacallao, Ketty; Monje, Paula V

    2013-01-01

    In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.

  6. Biochemical and molecular characterization of three new variants of AmpC beta-lactamases from Morganella morganii.

    Science.gov (United States)

    Power, Pablo; Galleni, Moreno; Ayala, Juan A; Gutkind, Gabriel

    2006-03-01

    Morganella morganii produces an inducible, chromosomally encoded AmpC beta-lactamase. We describe in this study three new variants of AmpC within this species with apparent pIs of 6.6 (M19 from M. morganii strain PP19), 7.4 (M29 from M. morganii strain PP29), and 7.8 (M37 from M. morganii strain PP37). After gene sequencing, deduced amino acid sequences displayed one to six substitutions when compared to the available Morganella AmpC sequences. An AmpR-encoding gene was also found upstream of ampC, including the LysR regulators' helix-turn-helix DNA-binding domain and the putative T-N11-A-protected region in the ampR-ampC intercistronic sequence. All three AmpC variants were purified from in vitro-generated derepressed mutants and showed overall similar kinetic parameters. None of the observed amino acid changes, occurring at the surface of the protein, appear to have a major influence in their catalytic properties. Morganella AmpCs exhibit the highest catalytic efficiencies (k(cat)/K(m)) on classical penicillins, cefoxitin, narrow-spectrum cephalosporins, and cefotaxime. Cefotaxime was more effectively hydrolyzed than other oxyimino-cephalosporins, whereas cefepime was 3 log-fold less efficiently hydrolyzed than other cephalosporins such as cephalothin. Several differences with other AmpC beta-lactamases were found. Ampicillin was more efficiently hydrolyzed than benzylpenicillin. High k(cat)/K(m) values were observed for oxacillin and piperacillin, which are usually poor substrates for AmpC. A fairly efficient hydrolysis of imipenem was detected as well. Aztreonam, carbenicillin, and tazobactam were effective transient inactivators of these variants.

  7. Effects of AMP579 and adenosine on L-type Ca2+ current in isolated rat ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Xiong WANG; Bo-wei WU; Dong-mei WU

    2005-01-01

    Aim: To compare the effects of AMP579 and adenosine on L-type Ca2+ current (ICa- L) in rat ventricular myocytes and explore the mechanism by which AMP579 acts on ICa-L. Methods: ICa-L was recorded by patch-clamp technique in whole-cell configuration. Results: Adenosine (10 nmol/L to 50 μmol/L) showed no effect on basal ICa- L, but it inhibited the ICa-L induced by isoproterenol 10 nmol/L in a concen tration-dependent manner with the IC50 of 13.06 μmol/L. Similar to adenosine,AMP579 also showed an inhibitory effect on the ICa-L induced by isoproterenol.AMP579 and adenosine (both in 10 μmol/L) suppressed isoproterenol-induced ICa-L by 11.1% and 5.2%, respectively. In addition, AMP579 had a direct inhibitory effect on basal ICa-L in a concentration-dependent manner with IC50 (1.17 μmol/L).PD116948 (30 μmol/L), an adenosine A1 receptor blocker, showed no action on the inhibitory effect of AMP579 on basal ICa-L. However, GF109203X (0.4 μmol/L), a special protein kinase C (PKC) blocker, could abolish the inhibitory effect of AMP579 on basal ICa-L. So the inhibitory effect of AMP579 on basal ICa-L was induced through activating PKC, but not linked to adenosine A1 receptor. Conclusion:AMP579 shows a stronger inhibitory effect than adenosine on the ICa-L induced by isoproterenol. AMP579 also has a strong inhibitory effect on basal ICa-L in rat ventricular myocytes. Activation of PKC is involved in the inhibitory effect of AMP579 on basal ICa-L at downstream-mechanism.

  8. Regulatory Action of Calcium Ion on Cyclic AMP-Enhanced Expression of Implantation-Related Factors in Human Endometrial Cells.

    Directory of Open Access Journals (Sweden)

    Kazuya Kusama

    Full Text Available Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP, but the role of intracellular calcium ion (Ca2+ on cAMP mediated-signaling in human endometrial stroma and glandular epithelia has not been well-characterized. The present study was designed to investigate the role of intracellular Ca2+ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP1 in human endometrial stromal cells (ESCs, and cyclooxygenase 2 (COX2 and prostaglandin E2 (PGE2 and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca2+ influx into ESCs with Ca2+ ionophores (alamethicin, ionomycin in a dose-dependent manner. Conversely, inhibitors of Ca2+ influx through L-type voltage-dependent Ca2+ channel (VDCC, nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca2+ release from the intracellular Ca2+ store, up-regulated prolactin and IGFBP-1 expression. Ca2+ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca2+ concentrations. Thus, a marked decrease in cytosolic Ca2+ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca2+ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells.

  9. Regulatory Action of Calcium Ion on Cyclic AMP-Enhanced Expression of Implantation-Related Factors in Human Endometrial Cells.

    Science.gov (United States)

    Kusama, Kazuya; Yoshie, Mikihiro; Tamura, Kazuhiro; Imakawa, Kazuhiko; Isaka, Keiichi; Tachikawa, Eiichi

    2015-01-01

    Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP), but the role of intracellular calcium ion (Ca2+) on cAMP mediated-signaling in human endometrial stroma and glandular epithelia has not been well-characterized. The present study was designed to investigate the role of intracellular Ca2+ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP)1 in human endometrial stromal cells (ESCs), and cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca2+ influx into ESCs with Ca2+ ionophores (alamethicin, ionomycin) in a dose-dependent manner. Conversely, inhibitors of Ca2+ influx through L-type voltage-dependent Ca2+ channel (VDCC), nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca2+ release from the intracellular Ca2+ store, up-regulated prolactin and IGFBP-1 expression. Ca2+ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca2+ concentrations. Thus, a marked decrease in cytosolic Ca2+ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca2+ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells. PMID:26161798

  10. An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

    Science.gov (United States)

    Huynh, TuAnh Ngoc; Luo, Shukun; Pensinger, Daniel; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2015-01-01

    The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network. PMID:25583510

  11. Investigation of Extended Spectrum β-lactamase and AmpC Enzyme in Enterobactericaeae%肠杆菌科细菌产AmpC酶及超广谱β-内酰胺酶的研究

    Institute of Scientific and Technical Information of China (English)

    王艾琳; 李坚; 张维瑜; 宿立娟

    2005-01-01

    目的探讨临床分离的耐药性肠杆菌科细菌产AmpCβ-内酰胺酶(AmpC酶)和超广谱β-内酰胺酶(ESBLs)的情况,为临床用药提供参考依据.方法采用纸片扩散确证试验检测ESBLs,三维试验法检测AmpC酶.结果207株肠杆菌科细菌中产ESBLs者95株(占45.9%),产AmpC酶者52株(占25.1%).结论 AmpC酶和ESBLs是导致肠杆菌科细菌耐药的两类主要酶.

  12. Relationship between Adaptation of the Folic Acid and the cAMP Mediated cGMP Response in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1983-01-01

    Chemotactic stimulation of post-vegetative Dictyostelium cells with folic acid or aggregative cells with cAMP results in a fast transient cGMP response which peaks at 10 s; basal levels are recovered in about 30-40 s. Stimulation with folic acid or cAMP rapidly desensitizes the cells for equal or lo

  13. Responsiveness of glycogen breakdown to cyclic AMP in perfused liver from rats with insulin-induced hypoglycemia

    Directory of Open Access Journals (Sweden)

    M. Vardanega-Peicher

    2003-01-01

    Full Text Available The responsiveness of glycogen breakdown to cAMP was investigated in isolated perfused liver from male Wistar fed rats (200-220 g with insulin-induced hypoglycemia. The activation of glycogenolysis by 3 µM cAMP was decreased (P<0.05 in livers from rats with hypoglycemia induced by the administration of insulin or during the direct infusion of insulin into the isolated liver. The direct effect of insulin on glycogen catabolism promoted by 3 µM cAMP occurred as early as 3 min after starting insulin infusion. In contrast, the cAMP agonists resistant to phosphodiesterases, 8Br-cAMP and 6MB-cAMP, used at the same concentration as cAMP, i.e., 3 µM, did not modify the effect of insulin. The data suggest that the decreased hepatic responsiveness of glycogen breakdown during insulin-induced hypoglycemia is a direct effect of insulin decreasing the intracellular levels of cAMP.

  14. 78 FR 1252 - CalAmp Wireless Networks Corporation (CWNC), Satellite Products Division, Including On-Site...

    Science.gov (United States)

    2013-01-08

    ... December 13, 2011 (76 FR 77556). At the request of the State of Minnesota, the Department reviewed the... Employment and Training Administration CalAmp Wireless Networks Corporation (CWNC), Satellite Products Division, Including On-Site Leased Workers From Select Staffing, Oxnard, CA; CalAmp Wireless...

  15. High prevalence of fecal carriage of extended spectrum beta-lactamase/AmpC-producing Enterobacteriaceae in cats and dogs

    NARCIS (Netherlands)

    Hordijk, J.; Schoormans, A.; Kwakernaak, M.; Duim, B.; Broens, E.; Dierikx, C.M.; Mevius, D.J.; Wagenaar, J.A.

    2013-01-01

    Extended-spectrum-ß-lactamase (ESBL)/AmpC producing Enterobacteriaceae have been reported worldwide amongst isolates obtained from humans, food-producing animals, companion animals, and environmental sources. However, data on prevalence of fecal carriage of ESBL/AmpC producing Enterobacteriaceae in

  16. Human cellular differences in cAMP - CREB signaling correlate with light-dependent melatonin suppression and bipolar disorder

    NARCIS (Netherlands)

    Gaspar, Ludmila; van de Werken, Maan; Johansson, Anne-Sophie; Moriggi, Ermanno; Owe-Larsson, Bjorn; Kocks, Janwillem W. H.; Lundkvist, Gabriella B.; Gordijn, Marijke C. M.; Brown, Steven A.

    2014-01-01

    Various lines of evidence suggest a mechanistic role for altered cAMP-CREB (cAMP response element - binding protein) signaling in depressive and affective disorders. However, the establishment and validation of human inter-individual differences in this and other major signaling pathways has proven

  17. Disease-tolerance of transgenic tobacco plants expressing Ah-AMP gene of Amaranthus hypochondriacus

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An antimicrobial peptide gene from Amaranthus hypochondriacus, Ah-AMP, was amplified by PCR and cloned. Sequence analysis results revealed that this gene is 261 bp in length encoding a precursor polypeptide of 87 amino acid residues. Ah-AMP gene was inserted in the binary vector pBin438 to construct a plant expression vector pBinAH916. Leave explants of Nicotiana tabacum var. SR1 were transformed with Agrobacterium tumefaciens LBA4404 harboring the above expression vector. Results from PCR, Southern and Northern blot analyses confirmed that the Ah-AMP gene had been integrated into the tobacco genome and was transcribed at mRNA level. Two bacterial-resistant transgenic plants were selected by inoculating the plants with Pseudomonas solanacearum and statistic analysis of two T1 lines showed that the resistance increased by 2.24 and 1.62 grade and the disease index decreased by 49.6% and 37.3% respectively when compared with the non-transformed control plants SR1. The results from challenging the plants with inoculums of Phytophthora parasitica showed that the symptom development was delayed and disease index was significantly reduced. These results suggest that Ah-AMP gene may be a potentially valuable gene for genetic engineering of plant for disease-resistance.

  18. Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Gadella, T.W.J.; Visser, A.J.W.G.; Haastert, van P.J.M.

    2003-01-01

    The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in th

  19. cAMP/PKA Signaling Inhibits Osteogenic Differentiation and Bone Formation in Rodent Models

    NARCIS (Netherlands)

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; Klundert, van de Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; Blitterswijk, van Clemens; Boer, de Jan

    2009-01-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable

  20. cAMP receptor protein (CRP) downregulates Klebsiella pneumoniae nif promoters in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating s70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity of Klebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level of nif gene expression. Here we show that CRP can repress s54-dependent nif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA and nifU), in a cAMP dependent fashion, in closed related E. coli background. Sequence analysis of these nif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstream cis elements at the promoters. In addition, the crp gene of K. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that of E. coli. This suggests that CRP-cAMP-mediated inhibition on the nif promoters could be the reason for carbon effect on nitrogen fixation and thus has its physiological significance. A novel regulatory linkage between carbon metabolism and nitrogen fixation is proposed.

  1. Developmental regulation and evolution of cAMP signaling in Dictyostelium

    NARCIS (Netherlands)

    Álvarez-Curto, Elisa

    2007-01-01

    Through evolution the social amoebas have developed mechanisms to adapt to environmental changes and ensure survival. This thesis explores the evolutionary origins of cAMP signalling and regulation of developmental decisions in the model organism Dictyostelium discoideum. It also shows the first mol

  2. 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.

  3. Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology.

    Science.gov (United States)

    Métrich, Mélanie; Berthouze, Magali; Morel, Eric; Crozatier, Bertrand; Gomez, Ana Maria; Lezoualc'h, Frank

    2010-03-01

    Exchange proteins directly activated by cyclic AMP (Epac) were discovered 10 years ago as new sensors for the second messenger cyclic AMP (cAMP). Epac family, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2 and function independently of protein kinase A. Given the importance of cAMP in the cardiovascular system, numerous molecular and cellular studies using specific Epac agonists have analyzed the role and the regulation of Epac proteins in cardiovascular physiology and pathophysiology. The specific functions of Epac proteins may depend upon their microcellular environments as well as their expression and localization. This review discusses recent data showing the involvement of Epac in vascular cell migration, endothelial permeability, and inflammation through specific signaling pathways. In addition, we present evidence that Epac regulates the activity of various cellular compartments of the cardiac myocyte and influences calcium handling and excitation-contraction coupling. The potential role of Epac in cardiovascular disorders such as cardiac hypertrophy and remodeling is also discussed.

  4. Role of 5'AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Wojtaszewski, Jørgen F. P.

    2008-01-01

    5'AMP-activated protein kinase (AMPK) is recognized as an important intracellular energy sensor, shutting down energy-consuming processes and turning on energy-generating processes. Discovery of target proteins of AMPK has dramatically increased in the past 10 years. Historically, AMPK was first...

  5. Exercise in rats does not alter hypothalamic AMP-activated protein kinase activity

    DEFF Research Database (Denmark)

    Andersson, Ulrika; Treebak, Jonas Thue; Nielsen, Jakob Nis;

    2005-01-01

    Recent studies have demonstrated that AMP-activated protein kinase (AMPK) in the hypothalamus is involved in the regulation of food intake. Because exercise is known to influence appetite and cause substrate depletion, it may also influence AMPK in the hypothalamus. Male rats that either rested...

  6. Synapse number and synaptic efficacy are regulated by presynaptic cAMP and protein kinase A.

    Science.gov (United States)

    Munno, David W; Prince, David J; Syed, Naweed I

    2003-05-15

    The mechanisms by which neurons regulate the number and strength of synapses during development and synaptic plasticity have not yet been defined fully. This lack of fundamental knowledge in the fields of neurodevelopment and synaptic plasticity can be attributed, in part, to compensatory mechanisms by which neurons accommodate for the loss of function in their synaptic partners. This is generally achieved either by scaling up neuronal transmitter release capabilities or by enhancing the postsynaptic responsiveness. Here, we demonstrate that regulation of synaptic strength and number between identified Lymnaea neurons visceral dorsal 4 (VD4, the presynaptic cell) and left pedal dorsal 1 (LPeD1, the postsynaptic cell) requires presynaptic activation of a cAMP-PKA-dependent signal. Experimental activation of the cAMP-PKA pathway resulted in reduced synaptic efficacy, whereas inhibition of the cAMP-PKA cascade permitted hyperinnervation and an overall enhancement of synaptic strength. Because synaptic transmission between VD4 and LPeD1 does not require a cAMP-PKA pathway, our data show that these messengers may play a novel role in regulating the synaptic efficacy during early synaptogenesis and plasticity.

  7. Cyclic AMP regulates NCAM expression and phosphorylation in cultured mouse astrocytes

    DEFF Research Database (Denmark)

    Gegelashvili, George; Andersson, A M; Schousboe, Arne;

    1993-01-01

    The neural cell adhesion molecule (NCAM) plays a key morphoregulatory role during neural development. Patterns of NCAM expression were investigated in cultured astrocytes exhibiting distinct states of morphological differentiation. In vitro differentiation of confluent primary cultures of astrocy......The neural cell adhesion molecule (NCAM) plays a key morphoregulatory role during neural development. Patterns of NCAM expression were investigated in cultured astrocytes exhibiting distinct states of morphological differentiation. In vitro differentiation of confluent primary cultures...... of astrocytes was enhanced by a long-term (7 days) treatment with dibutyryl cyclic AMP (dBcAMP). The diversity of NCAM mRNAs arising from alternative splicing of a single primary transcript was analyzed by Northern blotting. Synthetic DNA-oligonucleotide probes, designed to recognize selected exons (exons 7, 15......, and VASE) or exon combinations (exons 7/8, exons 12/13, and exons 12/a/AAG/13), revealed NCAM mRNA classes of 6.7, 5.2, and 2.9 kilobases (kb) in both control and dBcAMP-treated astrocytes. Although long-term treatment of astrocytes with dBcAMP did not change the qualitative pattern of NCAM transcripts...

  8. A high-speed CMOS current op amp for very low supply voltage operation

    DEFF Research Database (Denmark)

    Bruun, Erik

    1994-01-01

    , low voltage operation normally associated with current mode analog signal processing, the op amp has been designed to operate off a supply voltage of 1.5 V, and the signal path has been confined to N-channel transistors. With this design, a gain of 94 dB and a gain-bandwidth product of 65 MHz has been...

  9. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer;

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

  10. Detection of AmpC β-lactamase and drug resistance of Enterobacter cloacae

    Institute of Scientific and Technical Information of China (English)

    Rong WANG; Shangwei WU; Xue LI; Ping HE; Yunde LIU

    2009-01-01

    In order to provide useful information for effective control and clinical therapy of infection, the resistance status and the rate of carrying AmpC β-1actamase of Enterobacter cloacae (E. cloacae) were investigated. By VITEK (Bacterial automatic biochemical analyzer), the isolates of E. cloacae were identified and the drag resistance was measured. The AmpC enzyme was detected by the five-disk diffusion test. Antibiotic sensitivity test showed that the resistance effects of E. cloacae to cefazolin, cefoxitin and ampicillin were more serious, with resistant rates of 80.5%, 75.3% and 70.1%, respectively. However, it was more sensitive to Sulperazone (cefoperazone/sulbactam, 13.0%), amikacin (16.9%) and ciprofloxacin (19.5%). Meanwhile, the phenotype detection showed that 35.06% (27/77) isolates of E. cloacae produced AmpC β-1actamase. Most of E. cloacae are multi-drug resistant strains. Sulperazone (cefoperazone/sulbactam), a kind of component β-1actamase, is a more effective antibiotic for treating infection caused by E. cloacae. Unreasonable application of the third generation cephalosporins plays an important role in leading to emergence of high-yield AmpC β-1actamase strains, so antibiotics should be used wisely.

  11. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...

  12. 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

  13. Fast repair of dAMP hydroxyl radical adduct by verbascoside via electron transfer

    Institute of Scientific and Technical Information of China (English)

    石益民; 王文锋; 姚思德; 林维真; 韩镇辉; 师彦平; 贾忠建; 郑荣梁

    1999-01-01

    DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very impotant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells’ inadequate repair capacity. The repair activity and its mechanism of verbaseoside, isolated from Pedicularis species, towards dAMP-OH·was studied with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mmol/L dAMP aqueous solution containing verbascoside, the transient absorption spectrum of the hydroxyl adduct of dAMP decayed with the formation of that of the phenoxyl radical of verbascoside well under 100 microseconds after electron pulse irradiation. The result indicated that dAMP hydroxyl adducts can be repaired by verbascoside. The rate constants of the repair reaction was deduced to be 5.9×108 dm3·mol-1·s-1. A deeper understanding of this new repair mechanism will undo

  14. Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

    DEFF Research Database (Denmark)

    Hunter, Roger W; Treebak, Jonas Thue; Wojtaszewski, Jørgen;

    2011-01-01

    OBJECTIVE During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxicall...... and subsequent rise in cellular [G6P]....

  15. Experimental validation of a rate-based model for CO2 capture using an AMP solution

    DEFF Research Database (Denmark)

    Gabrielsen, Jostein; Svendsen, H. F.; Michelsen, Michael Locht;

    2007-01-01

    Detailed experimental data, including temperature profiles over the absorber, for a carbon dioxide (CO"2) absorber with structured packing in an integrated laboratory pilot plant using an aqueous 2-amino-2-methyl-1-propanol (AMP) solution are presented. The experimental gas-liquid material balance...

  16. A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Valentin-Hansen, Poul;

    2014-01-01

    The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid...

  17. 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.

  18. cAMP Stimulation of HCO3- Secretion Across Airway Epithelia

    Directory of Open Access Journals (Sweden)

    Welsh MJ

    2001-07-01

    Full Text Available To test for the presence of HCO(3(- transport across airway epithelia, we measured short-circuit current in primary cultures of canine and human airway epithelia bathed in a Cl(--free, HCO(3(-/CO(2-buffered solution. cAMP agonists stimulated a secretory current that was likely carried by HCO(3(- because it was absent in HCO(3(--free solutions. In addition, the cAMP-stimulated current was inhibited by the carbonic anhydrase inhibitor, acetazolamide, and by the apical addition of a blocker of cystic fibrosis transmembrane conductance regulator (CFTR, diphenylamine-2-carboxylate. The current was dependent on Na(+ because it was inhibited by removing Na(+ from the submucosal solution and by inhibition of the Na(+-K(+-ATPase with ouabain. The cAMP-stimulated current was absent in cystic fibrosis (CF airway epithelia. These data suggest that cAMP agonists can stimulate HCO(3(- secretion across airway epithelia and that CFTR may provide a conductive pathway for HCO(3(- movement across the apical membrane.

  19. Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Rikke Lind; Nielsen, Jesper Boye; Friis-Møller, Alice;

    2010-01-01

    OBJECTIVES: To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype. METHODS: Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporin...

  20. Forskolin and derivatives as tools for studying the role of cAMP.

    Science.gov (United States)

    Alasbahi, R H; Melzig, M F

    2012-01-01

    Forskolin (7beta-acetoxy-1alpha,6beta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one) is the first main labdane diterpenoid isolated from the roots of the Indian Plectranthus barbatus ANDREWS and one of the most extensively studied constituents of this plant. The unique character of forskolin as a general direct, rapid and reversible activator of adenylyl cyclase not only underlies its wide range of pharmacological effects but also renders it as a valuable tool in the study of the role of cAMP. The purpose of this review is to provide data presenting the utility of forskolin--as a cAMP activator--for studying the function of cAMP from different biological viewpoints as follows: 1) Investigation on the role of cAMP in various cellular processes in different organs such as gastrointestinal tract, respiratory tract, reproductive organs, endocrine system, urinary system, olfactory system, nervous system, platelet aggregating system, skin, bones, eyes, and smooth muscles. 2) Studies on the role of cAMP activation and inhibition to understand the pathogenesis (e.g. thyroid autoimmune disorders, leukocyte signal transduction defect in depression, acute malaria infection, secretory dysfunction in inflammatory diseases) as well as its possibly beneficial role for curing diseases such as the regulation of coronary microvascular NO production after heart failure, the attenuation of the development or progression of fibrosis in the heart and lungs, the augmentation of myo-protective effects of ischemic preconditioning especially in the failing hearts after myocardial infarction, the stimulation of the regeneration of injured retinal ganglion cells, the curing of glaucoma and inflammatory diseases, the reducing of cyst formation early in the polycystic kidney disease, and the management of autoimmune disorders by enhancing Fas-mediated apoptosis. 3) Studies on the role of cAMP in the mechanism of actions of a number of drugs and substances such as the effect of the

  1. Cyclic AMP-and beta-agonist-activated chloride conductance of a toad skin epithelium.

    Science.gov (United States)

    Willumsen, N J; Vestergaard, L; Larsen, E H

    1992-04-01

    1. The control by intracellular cyclic AMP and beta-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl(-)-sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM-amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/- 1 microA/cm2 (mean +/- S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was -96 +/- 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/- 0.003 (n = 54), and an intracellular Cl- activity of 40 +/- 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta-adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl- conductance identified by standard 36Cl- tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short-circuit current Isc = 0.1 +/- 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(-)-secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP- or isoprenaline-induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl- activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between -90 and -100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl- currents. 6. At V = -100 mV where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these

  2. Effects of an AMPS-modifi ed Polyacrylic Acid Superplasticizer on the Performance of Concrete Materials

    Institute of Scientific and Technical Information of China (English)

    CHEN Baofan

    2015-01-01

    A self-made 2-acrylamide-2-methyl propylene sulfonic (AMPS)-modified polyacrylic acid superplasticizer and two other commercially available superplasticizers with different molecular structures are used in this study to investigate the effect of an AMPS-modifi ed polyacrylic acid superplasticizer on the properties of concrete materials. In the experiments, initial and 1.5 h slumps over time after admixtion are determined by adding different dosages of three superplasticizers into the premixed concrete to characterize the slump loss resistance of the premixed concrete. The water-reducing rates of three different types of concrete are determined to characterize the water-reducing capacity of the concrete with each superplasticizer. The 3, 7 and 28 d compressive strength is determined to characterize the mechanical properties of the concrete with each superplasticizer. In the meanwhile, 1, 1.5 and 2.0 h slump loss rates over time after admixtion are determined by adding different dosages of the three superplasticizers into the high-performance concrete (HPC) to characterize the slump loss resistance of HPC. The 7, 28, 60 and 90 d compressive strength is determined to characterize the compressive properties of HPC with each superplasticizer. The dry shrinkage rates of three different types of HPC are determined with each superplasticizer. Electricfl ux after standard curing for 56 d and chloride ion diffusion coeffi cient after curing for 28 d of HPC are determined to characterize the impermeability of HPC with each superplasticizer. The cross-section was examined using a scanning electron microscopy (SEM) system. Results demonstrate that the AMPS-modifi ed polyacrylic acid superplasticizer has better water-reducing effect and slump than the two commercially available polyacrylic acid superplasticizers. The AMPS-modifi ed polyacrylic acid superplasticizer also shows signifi cant improvement of the compressive strength, especially in comprehensive performance of HPC. In

  3. FimL regulates cAMP synthesis in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Yuki F Inclan

    Full Text Available Pseudomonas aeruginosa, a ubiquitous bacteria found in diverse ecological niches, is an important cause of acute infections in immunocompromised individuals and chronic infections in patients with Cystic Fibrosis. One signaling molecule required for the coordinate regulation of virulence factors associated with acute infections is 3', 5'-cyclic adenosine monophosphate, (cAMP, which binds to and activates a catabolite repressor homolog, Vfr. Vfr controls the transcription of many virulence factors, including those associated with Type IV pili (TFP, the Type III secretion system (T3SS, the Type II secretion system, flagellar-mediated motility, and quorum sensing systems. We previously identified FimL, a protein with histidine phosphotransfer-like domains, as a regulator of Vfr-dependent processes, including TFP-dependent motility and T3SS function. In this study, we carried out genetic and physiologic studies to further define the mechanism of action of FimL. Through a genetic screen designed to identify suppressors of FimL, we found a putative cAMP-specific phosphodiesterase (CpdA, suggesting that FimL regulates cAMP levels. Inactivation of CpdA increases cAMP levels and restores TFP-dependent motility and T3SS function to fimL mutants, consistent with in vivo phosphodiesterase activity. By constructing combinations of double and triple mutants in the two adenylate cyclase genes (cyaA and cyaB, fimL, and cpdA, we show that ΔfimL mutants resemble ΔcyaB mutants in TM defects, decreased T3SS transcription, and decreased cAMP levels. Similar to some of the virulence factors that they regulate, we demonstrate that CyaB and FimL are polarly localized. These results reveal new complexities in the regulation of diverse virulence pathways associated with acute P. aeruginosa infections.

  4. Characterization of ESBL- and AmpC-Producing Enterobacteriaceae from Diseased Companion Animals in Europe.

    Science.gov (United States)

    Bogaerts, Pierre; Huang, Te-Din; Bouchahrouf, Warda; Bauraing, Caroline; Berhin, Catherine; El Garch, Farid; Glupczynski, Youri

    2015-12-01

    The study aimed to characterize beta-lactam resistance mechanisms of Enterobacteriaceae isolates recovered from diseased dogs and cats between 2008 and 2010 in a European surveillance program (ComPath I) for the antibiotic susceptibility of bacterial pathogens. A total of 608 non-duplicated Enterobacteriaceae isolates were obtained prior antibiotic treatment from diseased dogs (n=464) and cats (n=144). Among the 608 Enterobacteriaceae isolates, 22 presented a minimal inhibitory concentration against cefotaxime above EUCAST breakpoints of susceptibility. All the 22 isolates remained susceptible to carbapenems. Ten isolates were confirmed as extended-spectrum-beta-lactamase (ESBL) producers by PCR-sequencing of bla coding genes including 9 blaCTX-M (CTX-M-1, 14, 15, 32,…) and 1 blaTEM-52 and 12 were AmpC-producing isolates (10 plasmidic CMY-2 group and 2 isolates overexpressing their chromosomal AmpC). ESBLs and plasmid-mediated AmpC (pAmpC)-producing isolates were mainly recovered from dogs (n=17) suffering from urinary tract infections (n=13) and originated from eight different countries. ESBL-bearing plasmids were mostly associated with IncFII incompatibility groups while CMY-2 was predominantly associated with plasmid of the IncI1 group. ESBL/pAmpC-producing Escherichia coli belonged to phylogroup A (n=5), B2 (n=4), and D (n=5). Multilocus sequence typing analysis revealed that among three CTX-M-15-producing E. coli, two belong to sequence type (ST) 131 and one to ST405. The presence of CTX-M-15 including on IncFII plasmids in E. coli ST131-B2 has also been described in isolates of human origin. This suggests the possibility of exchanges of these isolates from humans to companion animals or vice-versa. PMID:26098354

  5. Compartmentalisation of cAMP-dependent signalling in blood platelets: The role of lipid rafts and actin polymerisation.

    Science.gov (United States)

    Raslan, Zaher; Naseem, Khalid M

    2015-01-01

    Prostacyclin (PGI2) inhibits blood platelets through the activation of membrane adenylyl cyclases (ACs) and cyclic adenosine 3',5'-monophosphate (cAMP)-mediated signalling. However, the molecular mechanism controlling cAMP signalling in blood platelet remains unclear, and in particular how individual isoforms of AC and protein kinase A (PKA) are coordinated to target distinct substrates in order to modulate platelet activation. In this study, we demonstrate that lipid rafts and the actin cytoskeleton may play a key role in regulating platelet responses to cAMP downstream of PGI2. Disruption of lipid rafts with methyl-beta-cyclodextrin (MβCD) increased platelet sensitivity to PGI2 and forskolin, a direct AC cyclase activator, resulting in greater inhibition of collagen-stimulated platelet aggregation. In contrast, platelet inhibition by the direct activator of PKA, 8-CPT-6-Phe-cAMP was unaffected by MβCD treatment. Consistent with the functional data, lipid raft disruption increased PGI2-stimulated cAMP formation and proximal PKA-mediated signalling events. Platelet inhibition, cAMP formation and phosphorylation of PKA substrates in response to PGI2 were also increased in the presence of cytochalasin D, indicating a role for actin cytoskeleton in signalling in response to PGI2. A potential role for lipid rafts in cAMP signalling is strengthened by our finding that a pool of ACV/VI and PKA was partitioned into lipid rafts. Our data demonstrate partial compartmentalisation of cAMP signalling machinery in platelets, where lipid rafts and the actin cytoskeleton regulate the inhibitory effects induced by PGI2. The increased platelet sensitivity to cAMP-elevating agents signalling upon raft and cytoskeleton disruption suggests that these compartments act to restrain basal cAMP signalling.

  6. Different roles of GNAS and cAMP signaling during early and late stages of osteogenic differentiation.

    Science.gov (United States)

    Zhang, S; Kaplan, F S; Shore, E M

    2012-09-01

    Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr. PMID:22903279

  7. IA/MA/AMPS 三元共聚物阻垢剂的性能评价%Performance evaluation of IA/MA/AMPS terpolymer scale inhibitor

    Institute of Scientific and Technical Information of China (English)

    张凤华; 刘鹏宇; 李飞

    2016-01-01

    In view of the IA/MA/AMPS terpolymer scale inhibitors.The effect of mass concentration of cal-cium ion,water temperature and pH of medium on the scale inhibition performance for calcium carbonate were investigated.The thermal stability of the copolymer scale inhibitors were analyzed via TG.The com-parison experiments of copolymer scale inhibitor and commercial scale inhibitor were carried on the disper-sion performance of scale inhibitors for ferric oxide.Results show that the ternary copolymer scale inhibitor has good thermal stability.The dispersion performance of scale inhibitors for ferric oxide is superior to com-mercial copolymer scale inhibitors WD-700 and T-225.It is found that copolymer scale inhibitors are suit-able for weak alkaline (pH =7 ~9),high temperature and moderate hardness water system.%针对实验合成的 IA /MA /AMPS 三元共聚物阻垢剂,分别考察了钙离子质量浓度、水样温度、水样 pH 值对共聚物阻垢性能的影响,合成的共聚物与市售阻垢剂分散氧化铁的性能进行了对比实验,并对提纯后的 IA /MA /AMPS 三元共聚物阻垢剂作 TG 分析。结果表明,合成的三元共聚物阻垢剂具有良好的热稳定性,分散氧化铁性能优于市售阻垢剂,适用于弱碱性、高温、中等矿化度的工业循环冷却水。

  8. The Application of Nanofiltration in the Extraction of jujube Cyclic Adenosinemonophosphate(cAMP)%纳膜过滤在提取红枣环磷酸腺苷(cAMP)中的应用

    Institute of Scientific and Technical Information of China (English)

    王春霞; 路福平; 刘逸寒; 杜连祥; 李明润

    2011-01-01

    Nanofiltration was used in the extraction of cAMP from Jujube.Jujube was first soaked , then through puree, enzyme hydrolysis, filtration, resin adsorption and formic acid elution.The results showed: Nanofiltration fraction have a peak at a wavelength of 258nm by UV which was the same as the cyclic adenosine monophosphate (cAMP)standard; the Nanofiltration discard part has no peak at 258nm; HPLC showed that Nanofiltration interception fraction has the same retention time as the cyclic adenosine monophosphate (cAMP) reference, while the Nanofiltration discard liquid do not have the peak at the same time point.This indicated that nanofiltration intercepted the target product of cyclic adenosine monophosphate (cAMP), successfully separated molecular , purified and concentrated cAMP.%红枣经过浸泡、打浆、酶解、压滤、树脂吸附、甲酸洗脱后,采用150 nm纳滤膜在压差为0.5~1 MPa下过滤,结果表明:纳滤截留组分经过紫外扫描在波长258 nm处峰值与环磷酸腺苷(cAMP)标准品相同,而纳滤液组分(弃去液)在波长258 nm处无峰;又经过HPLC检测纳滤截留组分与环磷酸腺苷(cAMP)标准品出峰时间相同,而纳滤液组分(弃去液)在此时间处无峰.表明纳滤截留目标产物环磷酸腺苷(cAMP)效果明显,分离了大分子组分及小分子组分,纳滤后利于浓缩、纯化得到环磷酸腺苷(cAMP)成品.

  9. Transcriptome changes and cAMP oscillations in an archaeal cell cycle

    Directory of Open Access Journals (Sweden)

    Soppa Jörg

    2007-06-01

    Full Text Available Abstract Background The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote. Results A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 μM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression. Conclusion The analysis of cell cycle-specific transcriptome changes of H. salinarum

  10. cAMP-dependent protein kinase A and the dynamics of epithelial cell surface domains : moving membranes to keep in shape

    NARCIS (Netherlands)

    Wojtal, Kacper A.; Hoekstra, Dick; van IJzendoorn, Sven C. D.

    2008-01-01

    Cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase A (PKA) are evolutionary conserved molecules with a well-established position in the complex network of signal transduction pathways. cAMP/PKA-mediated signaling pathways are implicated in many biological processes that cooperat

  11. Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

    DEFF Research Database (Denmark)

    Peraldi, P; Frödin, M; Barnier, J V;

    1995-01-01

    In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the r...

  12. Separation of effects of adenosine on energy metabolism from those on cyclic AMP in rat thymic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Nordeen, S.K.; Young, D.A.

    1977-08-10

    In rat thymic lymphocytes incubated for 2 h without exogenous energy-providing substrate, adenosine may be substituted for glucose as a means of maximally restoring energy metabolism and those cellular functions whose rates are sensitive to small changes in the energy balance, such as protein synthesis and uridine utilization for RNA synthesis. Since effects of adenosine in thymocytes and other cells have frequently been attributed to changes in cyclic AMP, this report investigates its possible involvement in these glucose-like restorative actions of adenosine. Although the same range of doses of adenosine effective at raising cyclic AMP also elicit roughly parallel stimulations of protein synthesis and uridine utilization, further results dissociate the restorative actions from those on cyclic AMP. (a) Other purine nucleosides mimic the glucose-like actions of adenosine without increasing cyclic AMP; (b) conversely, prostaglandin E/sub 1/ mimics the cyclic AMP response without restoring energy metabolism or energy-dependent functions; and (c) potentiation of the cyclic AMP response, either by inhibiting phosphodiesterase or adenosine deaminase, does not enhance the restorative response to a range of doses of adenosine. Finally, cyclic AMP-mediated glycogenolysis cannot account for the glucose-like effects since addition of adenosine increases, not decreases, levels of glycogen.

  13. REVIEW: Role of cyclic AMP signaling in the production and function of the incretin hormone glucagon-like peptide-1

    Science.gov (United States)

    Yu, Zhiwen; Jin, Tianru

    2008-01-01

    Pancreatic cells express the proglucagon gene (gcg) and thereby produce the peptide hormone glucagon, which stimulates hepatic glucose production and thereby increases blood glucose levels. The same gcg gene is also expressed in the intestinal endocrine L cells and certain neural cells in the brain. In the gut, gcg expression leads to the production of glucagon-like peptide-1 (GLP-1). This incretin hormone stimulates insulin secretion when blood glucose level is high. In addition, GLP-1 stimulates pancreatic cell proliferation, inhibits cell apoptosis, and has been utilized in the trans-differentiation of insulin producing cells. Today, a long-term effective GLP-1 receptor agonist has been developed as a drug in treating diabetes and potentially other metabolic disorders. Extensive investigations have shown that the expression of gcg and the production of GLP-1 can be activated by the elevation of the second messenger cyclic AMP (cAMP). Recent studies suggest that in addition to protein kinase A (PKA), exchange protein activated by cAMP (Epac), another effector of cAMP signaling, and the crosstalk between PKA and Wnt signaling pathway, are also involved in cAMP-stimulated gcg expression and GLP-1 production. Furthermore, functions of GLP-1 in pancreatic cells are mainly mediated by cAMP-PKA, cAMP-Epac and Wnt signaling pathways as well.

  14. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    Science.gov (United States)

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  15. Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter

    Science.gov (United States)

    Neasta, Jeremie; Fiorenza, Anna; He, Dao-Yao; Phamluong, Khanhky; Kiely, Patrick A.; Ron, Dorit

    2016-01-01

    RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription. PMID:27505161

  16. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia.

    Science.gov (United States)

    Ohnishi, Masatoshi; Urasaki, Tomoka; Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito; Inoue, Atsuko

    2015-11-13

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3', 5'-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction.

  17. AMP18 interacts with the anion exchanger SLC26A3 and enhances its expression in gastric cancer cells.

    Science.gov (United States)

    Di Stadio, Chiara Stella; Altieri, Filomena; Miselli, Giuseppina; Elce, Ausilia; Severino, Valeria; Chambery, Angela; Quagliariello, Vincenzo; Villano, Valentina; de Dominicis, Gianfranco; Rippa, Emilia; Arcari, Paolo

    2016-02-01

    AMP18 is a stomach-specific secreted protein expressed in normal gastric mucosa but absent in gastric cancer. AMP18 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids, present also in several unrelated proteins, and probably endowed with a chaperon-like activity. In this work, we exploited a functional proteomic strategy to identify potential AMP18 interactors with the aim to add knowledge on its functional role within gastric cell lines and tissues. To this purpose, recombinant biotinylated AMP18 was purified and incubated with protein extract from human normal gastric mucosa by applying an affinity chromatography strategy. The interacting proteins were identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. The pool of interacting proteins contained SLC26A3, a protein expressed in the apical membrane of intestinal epithelial cells, supposed to play a critical role in Cl(-) absorption and fluid homeostasis. The interaction was also confirmed by Western blot with anti-SLC26A3 on transfected AGS cell extract following AMP18 pull-down. Furthermore, the interaction between AMP18 and SLC26A3 was also validated by confocal microscopy that showed a co-localization of both proteins at plasma membrane level. More importantly, for the first time, we showed that SLC26A3 is down-regulated in gastric cancer and that the overexpression of AMP18 in AMP-transfected gastric cancer cells up-regulated the expression of SLC26A3 both at transcriptional and translational level, the latter probably through the activation of the MAP kinases pathway. These findings strongly suggest that AMP18 might play an anti-inflammatory role in maintaining mucosal integrity also by regulating SLC26A3 level. PMID:26700142

  18. Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    N O Yilmaz

    2013-01-01

    Full Text Available Background: Detecting plasmid-mediated AmpC (pAmpC β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82 and Escherichia coli (n: 191 were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX screening test, modified three dimensional test (M3DT, and multiplex polymerase chain reaction (PCR. Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.

  19. Assessment of Motor and Process Skills (AMPS)- arviointinemenetelmän käytettävyys : Systemaattinen kirjallisuuskatsaus

    OpenAIRE

    Hilli, Tanja

    2011-01-01

    Opinnäytteen tarkoituksena oli selvittää systemaattisen kirjallisuuskatsauksen avulla AMPS- arviointimenetelmän käytettävyyttä toimintaterapeutin ja asiakkaan näkökulmasta. Opinnäytteellä haluttiin selvittää AMPS- arviointimenetelmästä esille nousseita kokemuksia sekä menetelmän vaikutusta toimintaterapiaprosessiin. AMPS- arviointimenetelmästä on paljon tutkimuksia menetelmän luotettavuudesta ja toistettavuudesta, mikä tarkoittaa vahvaa näyttöön perustuvuutta. Opinnäytteessä haettiin tutkimuk...

  20. Biphasic regulation by dibutyryl cyclic AMP of tubulin and actin mRNA levels in neuroblastoma cells.

    OpenAIRE

    Ginzburg, I.; Rybak, S.; Kimhi, Y; Littauer, U. Z.

    1983-01-01

    Blot hybridization analysis that used labeled tubulin cDNA probes revealed that N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate [dibutyryl cyclic AMP (Bt2cAMP)] initially increases and later decreases the level of tubulin mRNA in a neuroblastoma-glioma hybrid cell line as well as in the parent cells. A significant increase in tubulin mRNA sequences is already evident 1 hr after the addition of Bt2cAMP to the neuroblastoma cells, and a maximal induction of 2-fold is seen after 12 hr. Cont...

  1. cAMP increases surface expression of NKCC2 in rat thick ascending limbs: role of VAMP.

    Science.gov (United States)

    Ortiz, Pablo A

    2006-03-01

    NaCl absorption by the thick ascending limb of Henle's loop (TAL) is mediated by the apical Na-K-2Cl cotransporter NKCC2. cAMP increases NaCl absorption in the TAL by stimulating NKCC2. In oocytes, cAMP increases NKCC2 activity by regulating its trafficking. However, the mechanism by which cAMP stimulates NKCC2 in TALs is not clear. We hypothesized that cAMP increases surface expression of NKCC2 and NaCl absorption in TALs and that vesicle-associated membrane protein (VAMP) is involved in this mechanism. We used surface biotinylation of rat medullary TALs (mTAL) to examine surface and total NKCC2 levels. When mTAL suspensions were treated with dibutyryl cAMP (db-cAMP) or forskolin plus IBMX for 20 min, surface NKCC2 expression increased by 126 +/- 23 and 92 +/- 17% above basal, respectively (P < 0.03). No changes in total NKCC2 expression were observed, suggesting that cAMP increased translocation of NKCC2. We studied the role of VAMP in NKCC2 translocation and found that incubating mTALs with tetanus toxin (30 nM), which inhibits vesicle trafficking by inactivating VAMP-2 and -3, completely blocked the stimulatory effect of db-cAMP on surface NKCC2 expression (tetanus toxin = 100% vs. tetanus toxin + db-cAMP = 102 +/- 21% of control; not significant). We studied VAMP-2 and -3 expression and localization in isolated perfused TALs by confocal microscopy and found that both of them were located in the subapical space of the TAL. Finally, in isolated perfused mTALs, db-cAMP increased net Cl absorption by 95.0 +/- 34.8% (P < 0.03), and pretreatment of TALs with tetanus toxin blocked the stimulation of Cl absorption (from 110.9 +/- 15.9 to 109.7 +/- 15.6 pmol.min(-1).mm(-1); not significant). We concluded that cAMP increases NKCC2 surface expression by a mechanism involving VAMP and that NKCC2 trafficking to the apical membrane is involved in the stimulation of TAL NaCl absorption by cAMP.

  2. Plasma levels of cAMP, cGMP and CGRP in sildenafil-induced headache

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Frandsen, E; Schifter, S;

    2004-01-01

    Sildenafil, a selective inhibitor of the cyclic guanosine monophosphate (cGMP) degrading phosphodiestrase 5 (PDE5), induced migraine without aura in 10 of 12 migraine patients and in healthy subjects it induced significantly more headache than placebo. The aim of the present study was to determine...... whether the pain-inducing effects of sildenafil would be reflected in plasma levels of important signalling molecules in migraine: cGMP, cyclic adenosine monophosphate (cAMP) and calcitonin gene-related peptide (CGRP). Ten healthy subjects (four women, six men) and 12 patients (12 women) suffering from...... migraine without aura were included in two separate double-blind, placebo-controlled, cross-over studies in which placebo or sildenafil 100 mg was administered orally. Plasma levels of CGRP, cAMP and cGMP were determined in blood from the antecubital vein. Despite the ability of sildenafil to induce...

  3. AMPS data management concepts. [Atmospheric, Magnetospheric and Plasma in Space experiment

    Science.gov (United States)

    Metzelaar, P. N.

    1975-01-01

    Five typical AMPS experiments were formulated to allow simulation studies to verify data management concepts. Design studies were conducted to analyze these experiments in terms of the applicable procedures, data processing and displaying functions. Design concepts for AMPS data management system are presented which permit both automatic repetitive measurement sequences and experimenter-controlled step-by-step procedures. Extensive use is made of a cathode ray tube display, the experimenters' alphanumeric keyboard, and the computer. The types of computer software required by the system and the possible choices of control and display procedures available to the experimenter are described for several examples. An electromagnetic wave transmission experiment illustrates the methods used to analyze data processing requirements.

  4. Addition of a gravity separation model to COBRA-IV for use in AMPS thermalhydraulics analysis

    International Nuclear Information System (INIS)

    The thermalhydraulic behaviour of the AMPS-1000 reactor is analyzed using the subchannel code COBRA-IV. The code was used to calculate the Critical Power Ratio (CPR) and the Minimum Departure from Nucleate Boiling (MDNBR) of the core. COBRA-IV was modified to allow for buoyancy, void diffusion, and void drift between adjacent subchannels. These modifications were verified against the sample case described in the ASSERT manual representing the calculated flow distribution in a 37-pin CANDU bundle. The predictions of ASSERT and COBRA of flow and enthalpy distribution across the bundle agree within 10%. CPR of the AMPS-1000 core was analyzed parametrically for submarine tilt angles of up to 90 degrees using the modified version of COBRA. The predicted change of CPR with tilt angle was found to be insignificant. (Author) 17 refs., 3 tabs., 8 figs

  5. Coexpression of ESBL, Amp C and MBL in gram negative bacilli

    Directory of Open Access Journals (Sweden)

    Ruturaj M. Kolhapure

    2015-10-01

    Conclusions: Klebsiella was the commonest isolate (28.47% followed by E coli (26.48%, Pseudomonas aeruginosa (19.54%, Enterobacter (8.92%, Acinetobacter (8.92% and Citrobacter (7.64%. A total of 272 out of 706 gram negative isolates were ESBL producers. ESBL production was seen more in E. coli followed by Klebsiella and P. aeruginosa. A total of 73 out of 706 isolates were inducible Amp C producers. AmpC production was seen more in Acinetobacter. A total of 65 out of 706 isolates were MBL producers. MBL Production was seen more in E. coli. [Int J Res Med Sci 2015; 3(10.000: 2698-2703

  6. Commissioning results of the Amsterdam Pulse Stretcher/Storage ring AmPS

    International Nuclear Information System (INIS)

    AmPS has been built to enhance substantially the main specifications of the 1 % duty factor 550 MeV electron accelerator facility MEA. The maximum energy will be raised to 0.9 GeV while the duty factor increases from 1 % to approximately 100 %. To this purpose the ring AmPS was added to the facility. Simultaneously the linac was upgraded both in current and energy. Two modes of operation for the ring are implemented: a Pulse Stretcher mode with 3 turn injection creating an external beam, and a Storage Mode with multi turn injection for internal target physics. The commissioning of the ring started in April 1992. Within two months 10 % duty factor beams could be delivered for electron scattering experiments. Meanwhile the performance of the machine has been improved dramatically. The actual performance of the ring is presented and is compared with the initial design goals

  7. A numerical method for the elliptic Monge-Amp\\`ere equation with transport boundary conditions

    CERN Document Server

    Froese, Brittany D

    2011-01-01

    The problem of optimal mass transport arises in numerous applications including image registration, mesh generation, reflector design, and astrophysics. One approach to solving this problem is via the Monge-Amp\\`ere equation. While recent years have seen much work in the development of numerical methods for solving this equation, very little has been done on the implementation of the transport boundary conditions. In this paper, we propose a method for solving the transport problem by iteratively solving a Monge-Amp\\`ere equation with Neumann boundary conditions. We present a new discretization for the equation, which converges to the viscosity solution. The resulting system is solved efficiently with Newton's method. We provide several challenging computational examples that demonstrate the effectiveness and efficiency ($O(M)-O(M^{1.3})$ time) of the proposed method.

  8. Fast finite difference solvers for singular solutions of the elliptic Monge-Amp\\'ere equation

    CERN Document Server

    Froese, Brittany D

    2010-01-01

    The elliptic Monge-Amp\\`ere equation is a fully nonlinear Partial Differential Equation which originated in geometric surface theory, and has been applied in dynamic meteorology, elasticity, geometric optics, image processing and image registration. Solutions can be singular, in which case standard numerical approaches fail. In this article we build a finite difference solver for the Monge-Amp\\'ere equation, which converges even for singular solutions. Regularity results are used to select a priori between a stable, provably convergent monotone discretization and an accurate finite difference discretization in different regions of the computational domain. This allows singular solutions to be computed using a stable method, and regular solutions to be computed more accurately. The resulting nonlinear equations are then solved by Newton's method. Computational results in two and three dimensions validate the claims of accuracy and solution speed. A computational example is presented which demonstrates the nece...

  9. Circadian regulation of Limulus visual functions: A role for octopamine and cAMP

    Directory of Open Access Journals (Sweden)

    Jasbir S. DALAL, Barbara-Anne BATTELLE

    2010-10-01

    Full Text Available The purpose of this contribution is to review our current understanding of the source and biochemistry of the circadian efferent input to the eyes of the American horseshoe crab Limulus polyphemus and the impact of this input on the structure, physiology and biochemistry of Limulus eyes. Special emphasis is given to the role of the biogenic amine octopamine and biochemical cascades it activates in the eyes. In addition to reviewing published data, we present new data showing that octopamine elevates cAMP levels in Limulus lateral eyes, and we partially characterize the pharmacology of the receptors involved in this response. We also present new data showing that octopamine regulates gene expression in Limulus lateral eyes by activating a cAMP cascade [Current Zoology 56 (5: 518–536, 2010].

  10. Associative conditioning analog selectively increases cAMP levels of tail sensory neurons in Aplysia.

    Science.gov (United States)

    Ocorr, K A; Walters, E T; Byrne, J H

    1985-04-01

    Bilateral clusters of sensory neurons in the pleural ganglia of Aplysia contain cells involved in a defensive tail withdrawal reflex. These cells exhibit heterosynaptic facilitation in response to noxious skin stimulation that can be mimicked by the application of serotonin. Recently it has been shown that this facilitation can be selectively amplified by the application of a classical conditioning procedure to individual sensory neurons. We now report that an analog of this classical conditioning paradigm produces a selective amplification of the cAMP content of isolated sensory neuron clusters. The enhancement is achieved within a single trial and appears to be localized to the sensory neurons. These results indicate that a pairing-specific enhancement of cAMP levels may be a biochemical mechanism for associative neuronal modifications and perhaps learning.

  11. Comment on ‘Didactical formulation of the Ampère law’

    Science.gov (United States)

    van Hees, Hendrik

    2014-09-01

    In this comment, we demonstrate that the example of a DC-conducting wire of finite length as an example for the application of the Biot-Savart law is flawed due to the non-conservation of electric charge for such an unphysical setting. The implications drawn in [1] on the restrictions of Ampère's circuital law in integral form are unnecessary as long as only physically realizable situations obeying the charge-conservation law are considered.

  12. Definition of Throw-Away Detectors (TADs) and VLF antenna for the AMPS laboratory

    Science.gov (United States)

    Koons, H. C.; Fennell, J. F.

    1975-01-01

    A Throw Away Detector (TAD)/subsatellite to be used as an experiment platform for the test flights to map the EMI from the shuttle and during the AMPS science flights is defined. A range of instrument platforms of varying capabilities is examined with emphasis on the EMI test vehicle. The operational support requirements of TAD/subsatellites are determined. The throw away detector is envisioned as a simple instrument package for supporting specific experiments.

  13. Age-related changes in AMP-activated protein kinase after stroke

    OpenAIRE

    Liu, Fudong; Benashski, Sharon E; Persky, Rebecca; Xu, Yan; Li, Jun; McCullough, Louise D.

    2011-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an evolutionary conserved energy sensor sensitive to changes in cellular AMP/ATP ratio which is activated by phosphorylation (pAMPK). pAMPK levels decrease in peripheral tissues with age, but whether this also occurs in the aged brain, and how this contributes to the ability of the aged brain to cope with ischemic stress is unknown. This study investigated the activation of AMPK and the response to AMPK inhibition after induced stroke...

  14. Berberine Promotes Glucose Consumption Independently of AMP-Activated Protein Kinase Activation

    OpenAIRE

    Miao Xu; Yuanyuan Xiao; Jun Yin; Wolin Hou; Xueying Yu; Li Shen; Fang Liu; Li Wei; Weiping Jia

    2014-01-01

    Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation wer...

  15. Simulation of Tunnel Junction in Cascade Solar Cell (GaAs/Ge) Using AMPS-1D

    OpenAIRE

    Benmoussa Dennai; H. Ben Slimane; Helmaoui, A.

    2014-01-01

    The development of the tunnel junction interconnect was key the first two-terminal monolithic, multi-junction solar cell development. This paper describes simulation for the tunnel junction (GaAs) between top cell (GaAs) and bottom cell (Ge). This solar cell cascade was simulated when using one dimensional simulation program called analysis of microelectronic and photonic structures (AMPS-1D). In the simulation, the thickness of the tunnel junction layer was varied from 10 to 50 nm. By varyin...

  16. Involvement of Hypothalamic AMP-Activated Protein Kinase in Leptin-Induced Sympathetic Nerve Activation

    OpenAIRE

    Mamoru Tanida; Naoki Yamamoto; Toshishige Shibamoto; Kamal Rahmouni

    2013-01-01

    In mammals, leptin released from the white adipose tissue acts on the central nervous system to control feeding behavior, cardiovascular function, and energy metabolism. Central leptin activates sympathetic nerves that innervate the kidney, adipose tissue, and some abdominal organs in rats. AMP-activated protein kinase (AMPK) is essential in the intracellular signaling pathway involving the activation of leptin receptors (ObRb). We investigated the potential of AMPKα2 in the sympathetic effec...

  17. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    OpenAIRE

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2013-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in...

  18. Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ionta, M. [Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas MG (Brazil); Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP (Brazil); Rosa, M.C.; Almeida, R.B.; Freitas, V.M.; Rezende-Teixeira, P.; Machado-Santelli, G.M. [Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP (Brazil)

    2012-05-25

    Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation [E-cadherin, connexin 26 (Cx26), and connexin 32 (Cx32)]. RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

  19. Transcriptional regulation induced by cAMP elevation in mouse Schwann cells

    OpenAIRE

    Schmid, Daniela; Zeis, Thomas; Schaeren-Wiemers, Nicole

    2014-01-01

    In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be mod...

  20. Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway

    OpenAIRE

    Almeida Parra, Ángeles; Moncada, Salvador; Bolaños Hernández, Juan Pedro

    2004-01-01

    El óxido nítrico activa la glucolisis en los astrocitos a través de una cascada de señalización en la que interviene la AMP kinasa, que fosforila (y activa) la fosfofructokinasa-2, enzima responsable de la biosíntesis de fructosa-2, 6-bisfosfato, efector alostérico positivo de la fosfofructokinasa-1. Este mecanismo es citoprotector.

  1. Computational prediction of cAMP receptor protein (CRP) binding sites in cyanobacterial genomes

    OpenAIRE

    Su Zhengchang; Xu Minli

    2009-01-01

    Abstract Background Cyclic AMP receptor protein (CRP), also known as catabolite gene activator protein (CAP), is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally st...

  2. Skeletal muscle metabolic flexibility : The roles of AMP-activated protein kinase and calcineurin

    OpenAIRE

    Long, Yun Chau

    2007-01-01

    Skeletal muscle fibers differ considerably in their metabolic and physiological properties. The metabolic properties of skeletal muscle display a high degree of flexibility which adapts to various physiological demands by shifting energy substrate metabolism. Studies were conducted to evaluate the roles of AMP-activated protein kinase (AMPK) and calcineurin in the regulation of skeletal muscle metabolism. Fasting elicited a coordinated expression of genes involved in lipid ...

  3. Occupational performance in school settings : evaluation and intervention using the school AMPS

    OpenAIRE

    Munkholm, Michaela

    2010-01-01

    Background: This thesis is was designed to evaluate aspects of reliability and validity of the School Version of the Assessment of Motor and Process Skills (School AMPS) (Fisher, Bryze, Hume, & Griswold, 2007), an observation-based evaluation of quality of occupational performance when children perform schoolwork tasks in school settings. The long term goal was to contribute to knowledge about children at risk or with mild disabilities who experience difficulties with occupational perform...

  4. Forskolin-free cAMP assay for Gi-coupled receptors.

    Science.gov (United States)

    Gilissen, Julie; Geubelle, Pierre; Dupuis, Nadine; Laschet, Céline; Pirotte, Bernard; Hanson, Julien

    2015-12-01

    G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands. PMID:26386312

  5. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

    Science.gov (United States)

    Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

    2015-10-01

    This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

  6. c-di-AMP Secreted by Intracellular Listeria monocytogenes Activates a Host Type I Interferon Response

    OpenAIRE

    Woodward, Joshua J.; Iavarone, Anthony T.; Daniel A Portnoy

    2010-01-01

    Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells. Induction of this host response is often dependent on microbial secretion systems, and in L. monocytogenes, is dependent on multi-drug efflux pumps (MDRs). Using L. monocytogenes mutants that over-expressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Over-expression of the di-adenylate cycla...

  7. Fructose-bisphosphatase as a substrate of cyclic AMP-dependent protein kinase.

    OpenAIRE

    Hosey, M M; Marcus, F

    1981-01-01

    We have tested rat liver fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) and three other gluconeogenic fructose-bisphosphatases as substrates for the catalytic subunit of cyclic AMP-dependent protein kinase. In contrast to the rat liver enzyme, homogeneous preparations of mouse liver, rabbit liver, and pig kidney fructose-bisphosphatase could not be phosphorylated by the kinase. Comparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the ...

  8. Macromodeling of the A.C. Characteristics of CMOS Op-Amps

    OpenAIRE

    Mandal, Pradip; Visvanathan, V

    1993-01-01

    An analytical-knowledge-based statistical method is developed to derive macromodels for the highly nonlinear AC response functions of CMOS op-amp circuits. Simple circuit analysis is used to motivate the use of logarithmic transformations. By this transformation, linear models are fitted over a large domain of the independent variables (typically, transistor widths) with very few circuit simulations. From circuit analysis it is deduced that poles and zeroes are appropriate for empirical model...

  9. Control of calcium transport in the myocardium by the cyclic AMP-Protein kinase system.

    Science.gov (United States)

    Katz, A M; Tada, M; Kirchberger, M A

    1975-01-01

    At least three mechanical changes characterize the response of cardiac muscle to agents that enhance cyclic AMP production. In common with other inotropic interventions, tension is augmented and the rate of tension rise is increased. The third response, acceleration of the rate of relaxation, is characteristic of the actions of beta-adrenergic agonists. These mechanical effects can be attributed to changes in (1) the amount of Ca2+ released during systole, (2) the rate of Ca2+ release at the onset of systole, and (3) the rate at which Ca2+ is reaccumulated by the sarcoplasmic reticulum at the end of systole. The ability of cyclic AMP-dependent protein kinases to phosphorylate the cardiac sarcoplasmic reticulum in vitro parallels stimulation of both Ca2+ transport and Ca2+-activated ATPase. The phosphoprotein formed in the presence of cyclic AMP and protein kinase has the chemical characteristics of a phosphoester, contains mostly phosphoserine, and has an electrophoretic mobility in SDS polyacrylamide gels that corresponds to a protein of 22,000 daltons. This 22,000-dalton protein, tentatively named phospholamban, thus differs from the acyl phosphooprotein formed by the Ca2+-transport ATPase, which as an apparent molecular weight of 90,000 to 100,000 daltons. Phospholamban has not been found in fast skeletal muscle, nor is Ca2+ transport accelerated by cyclic AMP and protein kinase in sarcoplasmic reticulum from these muslces which do not respond to beta-adrenergic agonists with accelerated relaxation. It thus appears likely that phosphorylation of phospholamban correlates both with an increased rate of Ca2+ transport by cardiac sarcoplasmic reticulum in vitro and accelerated relaxation in the intact myocardium. Preliminary findings are consistent with the view that phosphorylation of phospholamban may be related to other actions on Ca2+ fluxes brought about by agents which activate adenylate cyclase in the myocardium, but these interpretations must remain

  10. Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

    OpenAIRE

    Jong Hyun Kim; Ji-Man Park; Kyungmoo Yea; Hyun Wook Kim; Pann-Ghill Suh; Sung Ho Ryu

    2010-01-01

    BACKGROUND: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glu...

  11. Metabolic Basis for Thyroid Hormone Liver Preconditioning: Upregulation of AMP-Activated Protein Kinase Signaling

    OpenAIRE

    Videla, Luis A; Virginia Fernández; Pamela Cornejo; Romina Vargas

    2012-01-01

    The liver is a major organ responsible for most functions of cellular metabolism and a mediator between dietary and endogenous sources of energy for extrahepatic tissues. In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK) constitutes an intrahepatic energy sensor regulating physiological energy dynamics by limiting anabolism and stimulating catabolism, thus increasing ATP availability. This is achieved by mechanisms involving direct allosteric activation and reve...

  12. AutoAmp : An Open-Source Analog Amplifier Design Tool - For Classroom and Lab Purposes

    OpenAIRE

    Patri, Om Prasad; Rao, K. Sanmukh

    2012-01-01

    This correspondence presents an open-source tool AutoAmp developed at the Indian Institute of Technology, Guwahati. It is available at http://sourceforge.net/projects/autoamp-iitg/ This tool helps the user to design different types of electronic amplifiers, using solid state devices, for a given specification. It can handle several types of designs namely common-emitter BJT amplifier (single and two-stage), operational amplifiers (inverting and non-inverting) and power amplifier. Not only doe...

  13. 黏质沙雷菌中AmpC酶的检测及药敏率分析%Antimicrobial resistance of AmpC-producing serratia marcescens in Anhui

    Institute of Scientific and Technical Information of China (English)

    杨海飞; 程君; 胡立芬; 刘艳艳; 潘亚超; 朱玉林; 李家斌

    2012-01-01

    Objective To investigate the resistance of AmpC - producing Serratia marcescens for providing the scientific evidence in clinical diagnosis and treatment. Methods Potential AmpC - producing strains were detected by the cefoxitin disk diffusion method as described by CLSI 2010. Three - dimensional test was adopted for confirming AmpC - producing strains. The MICs of Serratia marcescens were determined by broth microdilution method. The results were judged according to the criteria recommended by CLSI 2010. Results The majority of Serratia marcescens were isolated from the specimen of sputum, accounting for 59. 6% . The bacteria were mostly detected in Respiratory department, followed by Intensive Care Unit, Gerontology Department. 41 of 104 isolates were identified as resistant to cefoxitin, accounting for 39. 4%. 8 strains ( 7. 7% ) produced AmpC β - lactamases. The antimicrobial susceptibility test showed that all strains were sensitive to imipenem and meropenem. The rates of resistance to cefepime, levofloxacin and gatifloxacin remained relatively unchanged between AmpC - producing strains and non - AmpC - producing strains. The resistant rates to other antimicrobial agents were significantly statistical difference ( P <0. 05 ) between the AmpC - producers and the non - AmpC - producers. Conclusion It showed that the production of AmpC β -lactamases in Serratia marcescens confers a high level of resistance to most kinds of antimicrobial agents. Carbapenems, fluoro-quinolones, and fourth generation cephalosporins should be selected in empirical therapy of serious infections caused by AmpC - producing Serratia marcescens.%目的 了解安徽省临床分离的104株黏质沙雷菌中AmpC酶的产生情况及其对常用抗菌药物的耐药特征,以指导临床合理用药.方法 采用头孢西丁纸片扩散法筛选疑产AmpC酶阳性菌株,并用酶粗提物进行三维试验确证产AmpC酶菌株.药敏试验采用琼脂稀释法,依据CLSI 2010年推荐的

  14. Cordycepin activates AMP-activated protein kinase (AMPK) via interaction with the γ1 subunit

    Science.gov (United States)

    Wu, Chongming; Guo, Yanshen; Su, Yan; Zhang, Xue; Luan, Hong; Zhang, Xiaopo; Zhu, Huixin; He, Huixia; Wang, Xiaoliang; Sun, Guibo; Sun, Xiaobo; Guo, Peng; Zhu, Ping

    2014-01-01

    Cordycepin is a bioactive component of the fungus Cordyceps militaris. Previously, we showed that cordycepin can alleviate hyperlipidemia through enhancing the phosphorylation of AMP-activated protein kinase (AMPK), but the mechanism of this stimulation is unknown. Here, we investigated the potential mechanisms of cordycepin-induced AMPK activation in HepG2 cells. Treatment with cordycepin largely reduced oleic acid (OA)-elicited intracellular lipid accumulation and increased AMPK activity in a dose-dependent manner. Cordycepin-induced AMPK activation was not accompanied by changes in either the intracellular levels of AMP or the AMP/ATP ratio, nor was it influenced by calmodulin-dependent protein kinase kinase (CaMKK) inhibition; however, this activation was significantly suppressed by liver kinase B1 (LKB1) knockdown. Molecular docking, fluorescent and circular dichroism measurements showed that cordycepin interacted with the γ1 subunit of AMPK. Knockdown of AMPKγ1 by siRNA substantially abolished the effects of cordycepin on AMPK activation and lipid regulation. The modulating effects of cordycepin on the mRNA levels of key lipid regulatory genes were also largely reversed when AMPKγ1 expression was inhibited. Together, these data suggest that cordycepin may inhibit intracellular lipid accumulation through activation of AMPK via interaction with the γ1 subunit. PMID:24286368

  15. Detection of AmpC β lactamases in gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Gunjan Gupta

    2014-01-01

    Full Text Available Amp C β-lactamases are clinically important cephalosporinases encoded on the chromosomes of many Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor/β-lactam combinations. The increase in antibiotic resistance among Gram-negative bacteria is a notable example of how bacteria can procure, maintain and express new genetic information that can confer resistance to one or several antibiotics. Detection of organisms producing these enzymes can be difficult, because their presence does not always produce a resistant phenotype on conventional disc diffusion or automated susceptibility testing methods. These enzymes are often associated with potentially fatal laboratory reports of false susceptibility to β-lactams phenotypically. With the world-wide increase in the occurrence, types and rate of dissemination of these enzymes, their early detection is critical. AmpC β-lactamases show tremendous variation in geographic distribution. Thus, their accurate detection and characterization are important from epidemiological, clinical, laboratory, and infection control point of view. This document describes the methods for detection for AmpC β-lactamases, which can be adopted by routine diagnostic laboratories.

  16. The 100,000 amp dc power supply for a staged hadron collider superferric magnet

    Energy Technology Data Exchange (ETDEWEB)

    Hays, Steven L.; Claypool, Bradley; Foster, G.William; /Fermilab

    2005-09-01

    A 1.5 volt 100,000 amp DC switcher power supply was developed for testing a superferric magnet string at FNAL. This supply was used during testing as both the ramping supply and holding supply powering a single magnet load with a total load resistance of 0.7{micro} Ohms. The supply consists of ten paralleled switcher cells, powered by a 400 volt/600 Amp DC power supply. Each cell consists of an IGBT H-bridge driving a step-down transformer at a switching frequency of 2 kHz. The transformer has an effective turns ratio of 224:1. The secondary consists of 32 parallel single-turn full wave rectifier windings. The rectification is done with 64 Shottky diodes. Each cell is rated at 1.5 volts/10,000 amps. During this test each cell was operated as a constant power source without load current or field feedback. This paper will describe the design of the switcher cell and control system used during testing. We will also describe the next level of improvements to the current feedback system to improve the ramp control.

  17. Mechanism of cAMP-induced H+ -efflux of Dictyostelium cells: a role for fatty acids

    Indian Academy of Sciences (India)

    H Flaadt; R Schaloske; D Malchow

    2000-09-01

    Aggregating Dictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H+ ATPase inhibitor concanamycin A and with the plasma membrane H+ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H+ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H+ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca2+-fluxes involves phospholipase A2 activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca2+-efflux. Here we show that arachidonic acid elicited H+-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca2+-relase channel.

  18. Preparation and Characteristics of Corn Straw-Co-AMPS-Co-AA Superabsorbent Hydrogel

    Directory of Open Access Journals (Sweden)

    Wei-Min Cheng

    2015-11-01

    Full Text Available In this study, the corn straw after removing the lignin was grafted with 2-acrylamido-2-methylpropanesulfonic acid (AMPS to prepare sulfonated cellulose. The grafting copolymerization between the sulfonated cellulose and acrylic acid (AA was performed using potassium persulfate and N,N′-methylenebisacrylamide as the initiator and crosslinking agent, respectively, to prepare corn straw-co-AMPS-co-AA hydrogels. The structure and properties of the resulting hydrogels were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, and dynamic rheometry. The effects of initiator, crosslinker, monomer neutralization degree, and temperature on the swelling ratio of the hydrogels were studied. The water retention, salt resistance, and recyclability of the corn straw-co-AMPS-co-AA hydrogels were also investigated. The optimum water absorptivity of the corn straw hydrogels was obtained at a polymerization temperature of 50 °C with 1.2% crosslinker, 1:7 ratio of the pretreated corn straw and AA, 2% initiator, and 50% neutralized AA.

  19. Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells.

    Science.gov (United States)

    Zakikhani, Mahvash; Dowling, Ryan; Fantus, I George; Sonenberg, Nahum; Pollak, Michael

    2006-11-01

    Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes, where it is often referred to as an "insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types, agents that facilitate signaling through these receptors would be expected to enhance proliferation. We show here that metformin acts as a growth inhibitor rather than an insulin sensitizer for epithelial cells. Breast cancer cells can be protected against metformin-induced growth inhibition by small interfering RNA against AMP kinase. This shows that AMP kinase pathway activation by metformin, recently shown to be necessary for metformin inhibition of gluconeogenesis in hepatocytes, is also involved in metformin-induced growth inhibition of epithelial cells. The growth inhibition was associated with decreased mammalian target of rapamycin and S6 kinase activation and a general decrease in mRNA translation. These results provide evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent population studies and justify further work to explore potential roles for activators of AMP kinase in cancer prevention and treatment. PMID:17062558

  20. Cross-talk between signaling pathways can generate robust oscillations in calcium and cAMP.

    Directory of Open Access Journals (Sweden)

    Fernando Siso-Nadal

    Full Text Available BACKGROUND: To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate 'bursting' oscillations of calcium and may enable better filtering of noise. CONCLUSION: We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.

  1. Seizure Suppression by High Temperature via cAMP Modulation in Drosophila

    Science.gov (United States)

    Saras, Arunesh; Tanouye, Mark A.

    2016-01-01

    Bang-sensitive (BS) Drosophila mutants display characteristic seizure-like activity (SLA) and paralysis after mechanical shock . After high-frequency electrical stimulation (HFS) of the brain, they generate robust seizures at very low threshold voltage. Here we report an important phenomenon, which effectively suppresses SLA in BS mutants. High temperature causes seizure suppression in all BS mutants (parabss1, eas, sda) examined in this study. This effect is fully reversible and flies show complete recovery from BS paralysis once the temperature effect is nullified. High temperature induces an increase in seizure threshold after a brief pulse of heat shock (HS). By genetic screening, we identified the involvement of cAMP in the suppression of seizures by high temperature. We propose that HS induces adenylyl cyclase which in turn increases cAMP concentration which eventually suppresses seizures in mutant flies. In summary, we describe an unusual phenomenon, where high temperature can suppress SLA in flies by modulating cAMP concentration. PMID:27558668

  2. Balanced phono-amps an extension to the 'the sound of silence' editions

    CERN Document Server

    Vogel, Burkhard

    2016-01-01

    In 12 chapters (Part I) this extension to the two 'The Sound of Silence' editions covers the development, calculation, construction and measurement of the fully differential (= balanced) phono-amp solution 'RIAA Phono-Amp Engine II'. Additionally, the balanced measurement amplifiers & measurement tools, the discussion on BJT gain stages, the 1/f noise calculation methods for BJTs, the calculation of fully-differential amplifiers, the numerous Mathcad worksheets, and the presentation of test and calibration records fill a further 10 chapters of Part II with essential knowhow that will equip the reader to develop his/her own phono-amp solution in the most balanced way - and of course, as low-noise as possible. Engine II offers eight different amplifying paths from the Engine's input to its output - solid-state as well as valve driven. To expand the input possibilities via an additional external input, any kind of other linear input amplifiers can be connected. Further, a selection of six highly diverse exte...

  3. Role of EPAC in cAMP-Mediated Actions in Adrenocortical Cells

    Science.gov (United States)

    Lewis, Aurélia E.; Aesoy, Reidun; Bakke, Marit

    2016-01-01

    Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology. PMID:27379015

  4. Numerical simulation of Ge solar cells using D-AMPS-1D code

    Energy Technology Data Exchange (ETDEWEB)

    Barrera, Marcela, E-mail: barrera@tandar.cnea.gov.ar [Comision Nacional de Energia Atomica, Avenida General Paz 1499, San Martin 1650, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) (Argentina); Rubinelli, Francisco [Instituto de Desarrollo Tecnologico para la Industria Quimica (INTEC)-CONICET, Gueemes 3450, Santa Fe 3000 (Argentina); Rey-Stolle, Ignacio [Instituto de Energia Solar, Universidad Politecnica de Madrid, Avenida Complutense 30, Madrid 28040 (Spain); Pla, Juan [Comision Nacional de Energia Atomica, Avenida General Paz 1499, San Martin 1650, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) (Argentina)

    2012-08-15

    A solar cell is a solid state device that converts the energy of sunlight directly into electricity by the photovoltaic effect. When light with photon energies greater than the band gap is absorbed by a semiconductor material, free electrons and free holes are generated by optical excitation in the material. The main characteristic of a photovoltaic device is the presence of internal electric field able to separate the free electrons and holes so they can pass out of the material to the external circuit before they recombine. Numerical simulation of photovoltaic devices plays a crucial role in their design, performance prediction, and comprehension of the fundamental phenomena ruling their operation. The electrical transport and the optical behavior of the solar cells discussed in this work were studied with the simulation code D-AMPS-1D. This software is an updated version of the one-dimensional (1D) simulation program Analysis of Microelectronic and Photonic Devices (AMPS) that was initially developed at The Penn State University, USA. Structures such as homojunctions, heterojunctions, multijunctions, etc., resulting from stacking layers of different materials can be studied by appropriately selecting characteristic parameters. In this work, examples of cells simulation made with D-AMPS-1D are shown. Particularly, results of Ge photovoltaic devices are presented. The role of the InGaP buffer on the device was studied. Moreover, a comparison of the simulated electrical parameters with experimental results was performed.

  5. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  6. Controlling fertilization and cAMP signaling in sperm by optogenetics.

    Science.gov (United States)

    Jansen, Vera; Alvarez, Luis; Balbach, Melanie; Strünker, Timo; Hegemann, Peter; Kaupp, U Benjamin; Wachten, Dagmar

    2015-01-20

    Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.

  7. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression.

    Science.gov (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  8. Analysis on drug resistance in Gram-negative bacilli producing AmpC enzyme%医院感染革兰阴性杆菌产AmpC酶状况及耐药性检测分析

    Institute of Scientific and Technical Information of China (English)

    李文波; 刘琼; 卢青云; 高武; 刘丽华; 王沛; 张玉娟

    2012-01-01

    Objective To investigate the nosocomial infection status and drug resistance of Gram-negative bacilli producing ceph-alosporinaseC AmpC enzyme). Methods Strains producing AmpC lactamases were detected by three-dimensional extract test. Drug susceptibility was determined by K - B disk diffusions method. Results The overall incidence rate of strains positive with AmpC lactamases was 18. 4% (109/331). The susceptibility rate of AmpC enzyme positive strains to third-generation cephalosporins, cephamycins,monobactam and antibiotics combined with inhibitors decreased,and relatively lower to Impenem,Cefepime and Ami-kacin, with resistace rate of (2/61) ,44. 26% (27/61) and 31. 1% (19/61) respectively. Resistance rates of AmpC enzyme positive strains were obviously greater than those of negative strains. Conclusion Drug resistance of Gram-negative bacilli might be associated with AmpC enzyme. Imipenem and forth-generation cephalosporins could be considered firstly for treatment of infection caused by AmpC enzyme positive Gram-negative bacilli.%目的 探讨产头孢菌素酶(AmpC酶)革兰阴性杆菌医院内感染现状及对药物敏感性的影响.方法 对临床标本进行分离鉴定,采用K-B法对常规药物进行耐药性检测,采用美国国家临床实验室标准化委员会(NCCLS)推荐的三维法检测AmpC酶.结果 在331株革兰阴性杆菌中检出产AmpC酶109株,产酶率为18.4%,产酶菌株对第3代头孢菌素、头霉素类、环丙沙星及含酶抑制剂复合物药物敏感率下降明显,对亚胺培南、头孢吡肟、丁胺卡那耐药率较低,分别为3.28%(2/61)、44.26%(27/61)、31.1%(19/61),产酶菌株对抗菌药物的耐药率明显高于非产酶菌株.结论 革兰阴性杆菌耐药与产AmpC酶有关,治疗该菌感染应选用亚胺培南、第4代头孢菌素等.

  9. Utility of the ceftazidime-imipenem antagonism test (CIAT to detect and confirm the presence of inducible AmpC beta-lactamases among enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    Vlademir Vicente Cantarelli

    2007-04-01

    Full Text Available Detection of AmpC beta-lactamase production by enterobacteria has been problematic. Contrary to ESBLs, no specific guidelines are available for detection and confirmation of AmpC production by clinical relevant microorganisms. Moreover, some bacterial species may produce inducible AmpC beta-lactamases that can be easily overlooked by routine susceptibility tests. We reported here a new test based on the strong inducible effect of imipenem on AmpC genes and the consequent antagonism with ceftazidime. This test is very simple and proved to be helpful in detecting AmpC-inducible enzymes among several species of clinical isolates.

  10. Constitutive high expression of chromosomal beta-lactamase in Pseudomonas aeruginosa caused by a new insertion sequence (IS1669) located in ampD

    DEFF Research Database (Denmark)

    Bagge, Niels; Ciofu, Oana; Hentzer, Morten;

    2002-01-01

    resistant, constitutive beta-lactamase-producing variant contained no mutations in ampD, but a point mutation was observed in ampR, resulting in an Asp-135-->Asn change. An identical mutation of AmpR in Enterobacter cloacae has been reported to cause a 450-fold higher AmpC expression. However, in many...... of the isolates expressing high levels of chromosomal beta-lactamase, no changes were found in either ampD, ampR, or in the promoter region of ampD, ampR, or ampC. Our results suggest that multiple pathways may exist leading to increased antimicrobial resistance due to chromosomal beta-lactamase.......The expression of chromosomal AmpC beta-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutations...

  11. Dibutyryl c-AMP as an inducer of sporidia formation: Biochemical and antigenic changes during morphological differentiation of Karnal bunt (Tilletia indica) pathogen in axenic culture

    Indian Academy of Sciences (India)

    Anil Kumar; Kaushlendra Tripathi; Manish Rana; Shalini Purwar; G K Garg

    2004-03-01

    Effect of dibutyryl adenosine 3′,5′-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation of Tilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages of T. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial anti-bodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase.

  12. The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

    DEFF Research Database (Denmark)

    Almblad, Henrik; Harrison, Joe J; Rybtke, Morten;

    2015-01-01

    infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors...... as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (c......AMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused...

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

    Science.gov (United States)

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

    2013-01-01

    Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in live corals followed a potential diel cycle, as they were higher during the day compared to the middle of the night. Coral homogenates exhibited some of the highest cAMP production rates ever to be recorded in any organism; this activity was inhibited by calcium ions and stimulated by bicarbonate. In contrast, zooxanthellae or mucus had >1000-fold lower AC activity. These results suggest that cAMP is an important regulator of coral physiology, especially in response to light, acid/base disturbances and inorganic carbon levels. PMID:23459251

  14. Twitching motility and cAMP levels: signal transduction through a single methyl-accepting chemotaxis protein.

    Science.gov (United States)

    Jansari, Vibhuti H; Potharla, Vishwakanth Y; Riddell, Geoff T; Bardy, Sonia L

    2016-06-01

    The Pseudomonas aeruginosa Chp chemosensory system regulates twitching motility, intracellular adenosine 3('') 5(')-cyclic monophosphate (cAMP) levels and is postulated to be involved in directional twitching towards phosphatidylethanolamine (PE). Because PilJ is the only methyl-accepting chemotaxis protein (MCP) identified in the Chp system, we determined the role of PilJ in mediating signal transduction for the distinct outputs of this system. Mutants that lack the periplasmic domain of PilJ (pilJΔ74-273) showed lower levels of cAMP but retained directional twitching towards PE. While initial studies revealed reduced twitching motility by PilJΔ74-273, this was due to decreased cAMP levels. Our data illustrate the importance of the periplasmic domain of PilJ in regulating cAMP. This is the first time a defined domain within PilJ has been identified as having a distinct role in signal transduction. PMID:27190147

  15. Cyclic AMP in female mouse brain is altered by the adrenocorticotropic hormone(4-9) analogue organon 2766.

    Science.gov (United States)

    Schneider, D R; Felt, B T; Murphy, S; Goldman, H

    1981-09-01

    Cyclic AMP content was determined in 12 brain regions of young adult female mice at 30 min and at 24 h following an intraperitoneal injection of the tri-substituted adrenocorticotropic hormone(4-9) [ACTH(4-9)] analogue Organon 2766 [ORG 2766]. Animals were killed by focused 3.5 kW microwave radiation applied for 350 ms. Unlike previously reported responses in male mice, at 30 min post-injection there were no detectable differences in cyclic AMP content between the placebo and ORG 2766-treated animals. By contrast, 24 h after injection, the content of cyclic AMP was changed significantly in 8 of the 12 brain regions examined: medulla-pons, septal area, thalamus, hypothalamus, hippocampus, olfactory bulb, and parietal and occipital cortices. In most of the regions examined, differences consisted of 50% or greater reductions of tissue cyclic AMP content. The changes were unrelated to the estrus cycle of these animals.

  16. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  17. Transcriptional regulation of 2',3'-cyclic nucleotide 3'-phosphodiesterase gene expression by cyclic AMP in C6 cells.

    Science.gov (United States)

    Gravel, M; Gao, E; Hervouet-Zeiber, C; Parsons, V; Braun, P E

    2000-11-01

    It was recently shown that the two transcripts encoding the isoforms of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP

  18. Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells

    Directory of Open Access Journals (Sweden)

    Neves-dos-Santos Sandra

    2010-01-01

    Full Text Available Abstract Background We investigated the effects of the signaling molecules, cyclic AMP (cAMP and protein-kinase C (PKC, on gap junctional intercellular communication (GJIC between thymic epithelial cells (TEC. Results Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. Conclusions Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

  19. Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp. hordei.

    Science.gov (United States)

    Hall, A A; Bindslev, L; Rouster, J; Rasmussen, S W; Oliver, R P; Gurr, S J

    1999-11-01

    Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew, is an obligate biotroph. On arrival on the host, a primary germ tube (PGT) emerges from the conidium. An appressorial germ tube (AGT) then appears, forms an appressorium, and effects host penetration. Such developmental precision may be due to multiple, plant-derived signals and to endogenous tactile and chemical signals. The transduction mechanism remains obscure. The isolation of an expressed sequence tag (EST) homologue of the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) enabled the corresponding gene to be characterized and the transcript to be identified in conidia and in PGT and AGT stage spores. cAMP-dependent PKA activity was detected in ungerminated conidia. These data suggest that PKA and cAMP are involved in conidial development. To substantiate this we exploited the responses of developing conidia to various surfaces, including exposure to the host leaf (fully inductive to AGT formation), cellulose membrane (semi-inductive), and glass (non-inductive). Assessment of fungal development, following application of exogenous cAMP or cAMP analogues, revealed that, at different concentrations and on different surfaces, cAMP either promoted or inhibited conidial differentiation. Various PKA inhibitors were tested for their effect on PKA activity and conidial development. A negative correlation was established between PKA inhibition in vitro and fungal development in vivo. Taken collectively, these data suggest that PKA and cAMP play a role in conidial differentiation in this obligate, plant-pathogenic fungus. PMID:10550894

  20. Strategies to overcome extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in shigellae

    OpenAIRE

    Livermore, David M.; Mushtaq, Shazad; Nguyen, Tue; Warner, Marina

    2011-01-01

    Abstract Oral cephalosporins and mecillinam are used to treat Shigella infections but are compromised by extended-spectrum ?-lactamases (ESBLs) and plasmid AmpC ?-lactamases. Potential solutions include combining an oral or intravenous cephalosporin with a ?-lactamase inhibitor (BLI) or using an oral penem. These strategies were examined using Escherichia coli transconjugants and clinical isolates with ESBLs or AmpC as a proxy for shigellae. The Clinical and Laboratory Standards In...

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

    OpenAIRE

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  3. Ca2+ participates in α1B-adrenoceptor-mediated cAMP response in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    Yao SONG; Yun-fang LI; Er-dan DONG; Qi-de HAN; You-yi ZHANG

    2005-01-01

    Aim: To investigate the α1B-adrenoceptor (α1B-AR)-mediated cAMP response and underlying mechanisms in HEK293 cells. Methods: Full-length cDNA encoding α1B-AR was transfected into HEK293 cells using the calcium phosphate precipitation method, and α1B-AR expression and cAMP accumulation were determined by using the saturation radioligand binding assay and ion-exchange chromatography, respectively. Results: Under agonist stimulation, α1B-AR mediated cAMP synthesis in HEK293 cells, and blockade by PLC-PKC or tyrosine kinase did not reduce cAMP accumulation induced by NE. Pretreatment with pertussis toxin(PTX) had little effect on basal cAMP accumulation as well as norepinephrine(NE)-stimulated cAMP accumulation. In addition, pretreatment with cholera toxin(CTX) neither mimicked nor blocked the effect induced by NE. The extracellular Ca2+ chelator egtazic acid (EGTA), nonselective Ca2+ channel blocker CdC12 and calmodulin (CaM) inhibitor W-7 significantly reduced NE-induced cAMP accumulation from 1.59%±0.47% to 1.00%±0.31%, 0.78%±0.23%, and 0.90%±0.40%,respectively. Conclusion: By coupling with a PTX-insensitive G protein, α1B-AR promotes Ca2+ influx via receptor-dependent Ca2+ channels, then Ca2+ is linked to CaM to form a Ca2+-CaM complex, which stimulates adenylyl cyclase (AC),thereby increasing the cAMP production in HEK293 cell lines.

  4. Exchange Protein Activated by cAMP Enhances Long-Term Memory Formation Independent of Protein Kinase A

    Science.gov (United States)

    Ma, Nan; Abel, Ted; Hernandez, Pepe J.

    2009-01-01

    It is well established that cAMP signaling within neurons plays a major role in the formation of long-term memories--signaling thought to proceed through protein kinase A (PKA). However, here we show that exchange protein activated by cAMP (Epac) is able to enhance the formation of long-term memory in the hippocampus and appears to do so…

  5. Glutamate Acts as a Key Signal Linking Glucose Metabolism to Incretin/cAMP Action to Amplify Insulin Secretion

    OpenAIRE

    Ghupurjan Gheni; Masahito Ogura; Masahiro Iwasaki; Norihide Yokoi; Kohtaro Minami; Yasumune Nakayama; Kazuo Harada; Benoit Hastoy; Xichen Wu; Harumi Takahashi; Kazushi Kimura; Toshiya Matsubara; Ritsuko Hoshikawa; Naoya Hatano; Kenji Sugawara

    2014-01-01

    Summary Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shu...

  6. Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

    Science.gov (United States)

    Witte, Chelsea E.; Whiteley, Aaron T.; Burke, Thomas P.; Sauer, John-Demian; Portnoy, Daniel A.; Woodward, Joshua J.

    2013-01-01

    ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. PMID:23716572

  7. CHARACTERIZATION OF P2-PURINOCEPTOR MEDIATED CYCLIC-AMP FORMATION IN MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1993-01-01

    1 The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2Cl2 myotubes. 2 ATP (100 muM) and ATPgammaS (100 muM) caused a sustained increase in cyclic AMP content of the cells,

  8. Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand

    Science.gov (United States)

    Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E.; Spurgeon, Harold A.; Sollott, Steven J.; Lakatta, Edward G.

    2011-01-01

    Rationale In sinoatrial node cells (SANC), Ca2+ activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca2+ cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca2+ oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca2+ to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Objective Since a negative feedback mechanism links ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca2+-cAMP/PKA dependent; and (2) due to its feed–forward nature, a decrease in flux through the Ca2+-cAMP/PKA signaling axis will reduce the basal ATP production rate. Methods and Results O2 consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca2+-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r2=0.96), and reduced O2 consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca2+ flux reduced the ATP level. Conclusions Feed-forward basal Ca2+-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca2+-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. PMID:21835182

  9. CA2+-DEPENDENT AND CA2+-INDEPENDENT MECHANISM OF CYCLIC-AMP REDUCTION - MEDIATION BY BRADYKININ B-2 RECEPTORS

    NARCIS (Netherlands)

    SIPMA, H; DENHERTOG, A; NELEMANS, A

    1995-01-01

    1 Bradykinin caused a transient reduction of about 25% in the cyclic AMP level in forskolin prestimulated DDT1 MF-2 smooth muscle cells (IC50: 36.4 +/- 4.9 nM) and a pronounced, sustained inhibition (40%) of the isoprenaline-stimulated cyclic AMP level (IC50: 37.5 +/- 1.1 nM). 2 The Ca2+ ionophore,

  10. A cAMP-independent carbohydrate-driven mechanism inhibits tnaA expression and TnaA enzyme activity in Escherichia coli.

    Science.gov (United States)

    Li, Gang; Young, Kevin D

    2014-09-01

    When Escherichia coli is grown in a medium lacking glucose or another preferred carbohydrate, the concentration of cAMP-cAMP receptor protein (cAMP-CRP) increases, and this latter complex regulates the expression of more than 180 genes. To respond rapidly to changes in carbohydrate availability, E. coli must maintain a suitable intracellular concentration of cAMP by either exporting or degrading excess cAMP. Currently, cAMP export via the TolC protein is thought to be more efficient at reducing these levels than is CpdA-mediated degradation of cAMP. Here, we compared the contributions of TolC and CpdA by measuring the expression of cAMP-regulated genes that encode tryptophanase (TnaA) and β-galactosidase. In the presence of exogenous cAMP, a tolC mutant produced intermediate levels of these enzymes, suggesting that cAMP levels were held in check by CpdA. Conversely, a cpdA mutant produced much higher amounts of these enzymes, indicating that CpdA was more efficient than TolC at reducing cAMP levels. Surprisingly, expression of the tnaA gene halted rapidly when glucose was added to cells lacking both TolC and CpdA, even though under these conditions cAMP could not be removed by either pathway and tnaA expression should have remained high. This result suggests the existence of an additional mechanism that eliminates intracellular cAMP or terminates expression of some cAMP-CRP-regulated genes. In addition, adding glucose and other carbohydrates rapidly inhibited the function of pre-formed TnaA, indicating that TnaA is regulated by a previously unknown carbohydrate-dependent post-translational mechanism.

  11. Timing is critical for effective glucocorticoid receptor mediated repression of the cAMP-induced CRH gene.

    Directory of Open Access Journals (Sweden)

    Siem van der Laan

    Full Text Available Glucocorticoid negative feedback of the hypothalamus-pituitary-adrenal axis is mediated in part by direct repression of gene transcription in glucocorticoid receptor (GR expressing cells. We have investigated the cross talk between the two main signaling pathways involved in activation and repression of corticotrophin releasing hormone (CRH mRNA expression: cyclic AMP (cAMP and GR. We report that in the At-T20 cell-line the glucocorticoid-mediated repression of the cAMP-induced human CRH proximal promoter activity depends on the relative timing of activation of both signaling pathways. Activation of the GR prior to or in conjunction with cAMP signaling results in an effective repression of the cAMP-induced transcription of the CRH gene. In contrast, activation of the GR 10 minutes after onset of cAMP treatment, results in a significant loss of GR-mediated repression. In addition, translocation of ligand-activated GR to the nucleus was found as early as 10 minutes after glucocorticoid treatment. Interestingly, while both signaling cascades counteract each other on the CRH proximal promoter, they synergize on a synthetic promoter containing 'positive' response elements. Since the order of activation of both signaling pathways may vary considerably in vivo, we conclude that a critical time-window exists for effective repression of the CRH gene by glucocorticoids.

  12. Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew.

    Science.gov (United States)

    Kinane, J; Dalvin, S; Bindslev, L; Hall, A; Gurr, S; Oliver, R

    2000-05-01

    Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner. PMID:10796015

  13. An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome.

    Science.gov (United States)

    Harrison, C J; Moorman, A V; Schwab, C; Carroll, A J; Raetz, E A; Devidas, M; Strehl, S; Nebral, K; Harbott, J; Teigler-Schlegel, A; Zimmerman, M; Dastuge, N; Baruchel, A; Soulier, J; Auclerc, M-F; Attarbaschi, A; Mann, G; Stark, B; Cazzaniga, G; Chilton, L; Vandenberghe, P; Forestier, E; Haltrich, I; Raimondi, S C; Parihar, M; Bourquin, J-P; Tchinda, J; Haferlach, C; Vora, A; Hunger, S P; Heerema, N A; Haas, O A

    2014-05-01

    Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL. PMID:24166298

  14. 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.

  15. Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides.

    Science.gov (United States)

    Bartlett, Rush L; Medow, Matthew R; Panitch, Alyssa; Seal, Brandon

    2012-04-01

    Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.

  16. Studies on the Correlation between Germination and cAMP Content of Different Color Chlamydospores in Ustiloginoidea virens

    Institute of Scientific and Technical Information of China (English)

    Xiaojuan LI; Erming LIU; Yuan LI; Zuohua REN; Lang YANG; Juanfang CHEN; Dandan HONG; Shuangqing LIU; Xinyu ZHOU; Minjie LIU

    2016-01-01

    Ustilaginoidea virens is the causal agent of a serious disease of rice. To reveal the relationship between germination and the 3'-5'-cyclic adenosine monophosphate (cAMP) content from the dormant (black or green-black) and non-dormant(yel ow) chlamydospore in Ustiloginoidea virens,this study adopts ultrasonic-bath method and high-performance liquid chromatography (HPLC) method, for extraction cAMP content of the different color chlamydospore. The results demonstrated that, as for the freshly chlamydospores col ected from the false smut bal s during their germination, cAMP content of yel ow chlamydospore ap-peared a slight growth during 0-12 h, and showed a rapid declining as the germination time extended (12-48 h) . Above al , in yel ow chlamydospores, the germination rate and the content of cAMP presented a very notably negative correlation (|r|=0.861 9>r0.01=0.834), but the correlation between germination rate and content of cAMP was not obvious in black chlamydospores. The germination rate and the content of cAMP presented a notably positive correlation under different storage period (0, 2, 4, 6, 8, 10 months) of the two color chlamydospores (the yel ow of chlamydospore: |r| = 0.785 1 > r0.05 = 0.707; the black of chlamy-dospore: |r| = 0.957 9 > r0.05 = 0.707).

  17. Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP).

    Science.gov (United States)

    Kim, Henna; Youn, Suk-Jun; Kim, Seong Ok; Ko, Junsang; Lee, Jie-Oh; Choi, Byong-Seok

    2015-06-26

    Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3',3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.

  18. Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes

    International Nuclear Information System (INIS)

    Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP) + 1 microM dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP + DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-14C]glutamate was recovered in the medium as radioactive glutamine, 2-3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate

  19. Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells.

    Science.gov (United States)

    Lee, Wei-Hwa; Lin, Ren-Jye; Lin, Shyr-Yi; Chen, Yu-Chien; Lin, Hsiu-Ming; Liang, Yu-Chih

    2011-12-28

    AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes. PMID:22098542

  20. ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independent pathways.

    Science.gov (United States)

    Carriba, Paulina; Pardo, Luis; Parra-Damas, Arnaldo; Lichtenstein, Mathieu P; Saura, Carlos A; Pujol, Aurora; Masgrau, Roser; Galea, Elena

    2012-09-01

    In neurons, it is well established that CREB contributes to learning and memory by orchestrating the translation of experience into the activity-dependent (i.e., driven by neurotransmitters) transcription of plasticity-related genes. The activity-dependent CREB-triggered transcription requires the concerted action of cyclic AMP/protein kinase A and Ca(2+) /calcineurin via the CREB-regulated transcription co-activator (CRTC). It is not known, however, whether a comparable molecular sequence occurs in astrocytes, despite the unquestionable contribution of these cells to brain plasticity. Here we sought to determine whether and how ATP and noradrenaline cause CREB-dependent transcription in rat cortical astrocyte cultures. Both transmitters induced CREB phosphorylation (Western Blots), CREB-dependent transcription (CRE-luciferase reporter assays), and the transcription of Bdnf, a canonical regulator of synaptic plasticity (quantitative RT-PCR). We indentified a Ca(2+) and diacylglycerol-independent protein kinase C at the uppermost position of the cascade leading to CREB-dependent transcription. Notably, CREB-dependent transcription was partially dependent on ERK1/2 and CRTC, but independent of cyclic AMP/protein kinase A or Ca(2+) /calcineurin. We conclude that ATP and noradrenaline activate CREB-dependent transcription in cortical astrocytes via an atypical protein kinase C. It is of relevance that the signaling involved be starkly different to the one described in neurons since there is no convergence of Ca(2+) and cyclic AMP-dependent pathways on CRTC, which, moreover, exerts a modulatory rather than a central role. Our data thus point to the existence of an alternative, non-neuronal, glia-based role of CREB in plasticity.

  1. Aclidinium bromide combined with formoterol inhibits remodeling parameters in lung epithelial cells through cAMP.

    Science.gov (United States)

    Lambers, Christopher; Costa, Luigi; Ying, Qi; Zhong, Jun; Lardinois, Didier; Dekan, Gerhard; Schuller, Elisabeth; Roth, Michael

    2015-12-01

    Combined muscarinic receptor antagonists and long acting β2-agonists improve symptom control in chronic obstructive pulmonary disease (COPD) significantly. In clinical studies aclidinium bromide achieved better beneficial effects than other bronchodilators; however, the underlying molecular mechanisms are unknown. This study assessed the effect of aclidinium bromide combined with formoterol on COPD lung (n=20) and non-COPD lung (n=10) derived epithelial cells stimulated with TGF-β1+carbachol on: (i) the generation of mesenchymal cells in relation to epithelial cells, (II) extracellular matrix (ECM) deposition, and (iii) the interaction of ECM on the generation of epithelial and mesenchymal cells. TGF-β1+carbachol enhanced the generation of mesenchymal cells, which was significantly reduced by aclidinium bromide or formoterol. The effect of combined drugs was additive. Inhibition of p38 MAP kinase and Smad by specific inhibitors or aclidinium bromide reduced the generation of mesenchymal cells. In mesenchymal cells, TGF-β1+carbachol induced the deposition of collagen-I and fibronectin which was prevented by both drugs dose-dependently. Formoterol alone reduced collagen-I deposition via cAMP, this however, was overruled by TGF-β1+carbachol and rescued by aclidinium bromide. Inhibition of fibronectin was cAMP independent, but involved p38 MAP kinase and Smad. Seeding epithelial cells on ECM collagen-I and fibronectin induced mesenchymal cell generation, which was reduced by aclidinium bromide and formoterol. Our results suggest that the beneficial effect of aclidinium bromide and formoterol involves cAMP affecting both, the accumulation of mesenchymal cells and ECM remodeling, which may explain the beneficial effect of the drugs on lung function in COPD. PMID:26546746

  2. Simulation of Tunnel Junction in Cascade Solar Cell (GaAs/Ge Using AMPS-1D

    Directory of Open Access Journals (Sweden)

    Benmoussa Dennai

    2014-11-01

    Full Text Available The development of the tunnel junction interconnect was key the first two-terminal monolithic, multi-junction solar cell development. This paper describes simulation for the tunnel junction (GaAs between top cell (GaAs and bottom cell (Ge. This solar cell cascade was simulated when using one dimensional simulation program called analysis of microelectronic and photonic structures (AMPS-1D. In the simulation, the thickness of the tunnel junction layer was varied from 10 to 50 nm. By varying thickness of tunnel junction layer the simulated device performance was demonstrate in the form of current-voltage(I-V characteristics and quantum efficiency (QE.

  3. Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

    OpenAIRE

    Shapiro, M.; Matthews, J.; Hecht, G; Delp, C; Madara, J. L.

    1991-01-01

    T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with h...

  4. Cefotaximase and AmpC-producing Shigella flexneri in case of dysentery from southern India

    Directory of Open Access Journals (Sweden)

    S Oommen

    2013-01-01

    Full Text Available Diarrhoea and dysentery caused by Shigella spp. are major public health concerns. Emerging multidrug resistance (MDR in this pathogen further complicates this disease. Extended spectrum β-lactamases (ESBLs have been described in this pathogen, which significantly compromises the treatment options for shigellosis. The usual ESBLs seen are sulfhydryl variable (SHV-type; cefotaximases (CTX-M are very uncommonly detected. Here, we report a CTX-M type and AmpC-producing Shigella flexneri from a three-year-old boy residing in Central Kerala, South India.

  5. Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates

    DEFF Research Database (Denmark)

    Hansen, Anna Mette; Bonke, Gitte; Larsen, Camilla Josephine;

    2016-01-01

    Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge....... In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59...

  6. Effects of metformin on expression of AMP-activated protein kinase in rat glomerular mesangial cells

    Institute of Scientific and Technical Information of China (English)

    顾俊菲

    2014-01-01

    Objective To observe the effects of metformin on expression of Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK),nuclear factor-κB(NF-κB)and transforming growth factorβ1(TGF-β1)in cultured rat glomerular mesangial cells(MCs),and explore its reno-protective mechanisms.Methods MCs were cultured in the medium with normal glucose(group NG,5.6mmol/L),high glucose(group HG,25 mmol/L)and different concentrations of metformin(group M1,M2,M3).After 48 h exposure,the supernatants and MCs

  7. AMP-activated protein kinase downregulates Kv7.1 cell surface expression

    DEFF Research Database (Denmark)

    Andersen, Martin N; Krzystanek, Katarzyna; Jespersen, Thomas;

    2012-01-01

    in response to polarization of the epithelial Madin-Darby canine kidney (MDCK) cell line and that this was mediated by activation of protein kinase C (PKC). In this study, the pathway downstream of PKC, which leads to internalization of Kv7.1 upon cell polarization, is elucidated. We show by confocal...... microscopy that Kv7.1 is endocytosed upon initiation of the polarization process and sent for degradation by the lysosomal pathway. The internalization could be mimicked by pharmacological activation of the AMP-activated protein kinase (AMPK) using three different AMPK activators. We demonstrate...

  8. High prevalence of fecal carriage of Extended Spectrum β-Lactamase/AmpC-producing Enterobacteriaceae in cats and dogs

    Directory of Open Access Journals (Sweden)

    Joost eHordijk

    2013-08-01

    Full Text Available ESBL/AmpC producing Enterobacteriaceae have been reported worldwide amongst isolates obtained from humans, food-producing animals, companion animals and environmental sources. However, data on prevalence of fecal carriage of ESBL/AmpC producing Enterobacteriaceae in healthy companion animals is limited. This pilot study describes the prevalence of ESBL/AmpC encoding genes in healthy cats and dogs, and cats and dogs with diarrhea. Twenty fecal samples of each group were cultured on MacConkey agar supplemented with 1 mg/L cefotaxime and in LB-enrichment broth supplemented with 1 mg/L cefotaxime, which was subsequently inoculated on MacConkey agar supplemented with 1 mg/L cefotaxime. ESBL/AmpC genes were identified using the Check-Points CT103 micro array kit and subsequently by sequencing analysis. Chromosomal ampC promoter mutations were detected by PCR and sequencing analysis. From the healthy and diarrheic dogs, respectively 45% and 55% were positive for E. coli with reduced susceptibility for cefotaxime. From the healthy and diarrheic cats, the estimated prevalence was respectively 0% and 25%. One diarrheic cat was positive for both reduced susceptible E. coli and P. mirabilis. The ESBL/AmpC genes found in this study were mainly blaCTX-M-1, but also blaCTX-M-14, blaCTX-M-15, blaTEM-52-StPaul, blaSHV-12 and blaCMY-2 were detected. This pilot study showed that the prevalence of ESBL/AmpC producing Enterobacteriaceae in healthy and diarrheic dogs, and diarrheic cats was relatively high. Furthermore the genes found were similar to those found in isolates of both human and food producing animal origin. However, since the size of this study was relatively small, extrapolation of the data to the general population of cats and dogs should be done with great care.

  9. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Directory of Open Access Journals (Sweden)

    Pawin Pongkorpsakol

    2014-09-01

    Full Text Available Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84 cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+-K(+ ATPases and Na(+-K(+-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+ channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+-activated basolateral K(+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  10. Interaction between cAMP and intracellular Ca(2+)-signaling pathways during odor-perception and adaptation in Drosophila.

    Science.gov (United States)

    Murmu, Meena Sriti; Martin, Jean-René

    2016-09-01

    Binding of an odorant to olfactory receptors triggers cascades of second messenger systems in olfactory receptor neurons (ORNs). Biochemical studies indicate that the transduction mechanism at ORNs is mediated by cyclic adenosine monophosphate (cAMP) and/or inositol,1,4,5-triphosphate (InsP3)-signaling pathways in an odorant-dependent manner. However, the interaction between these two second messenger systems during olfactory perception or adaptation processes is much less understood. Here, we used interfering-RNAi to disrupt the level of cAMP alone or in combination with the InsP3-signaling pathway cellular targets, InsP3 receptor (InsP3R) or ryanodine receptor (RyR) in ORNs, and quantify at ORN axon terminals in the antennal lobe, the odor-induced Ca(2+)-response. In-vivo functional bioluminescence Ca(2+)-imaging indicates that a single 5s application of an odor increased Ca(2+)-transients at ORN axon terminals. However, compared to wild-type controls, the magnitude and duration of ORN Ca(2+)-response was significantly diminished in cAMP-defective flies. In a behavioral assay, perception of odorants was defective in flies with a disrupted cAMP level suggesting that the ability of flies to correctly detect an odor depends on cAMP. Simultaneous disruption of cAMP level and InsP3R or RyR further diminished the magnitude and duration of ORN response to odorants and affected the flies' ability to detect an odor. In conclusion, this study provides functional evidence that cAMP and InsP3-signaling pathways act in synergy to mediate odor processing within the ORN axon terminals, which is encoded in the magnitude and duration of ORN response. PMID:27212269

  11. The camp analogue, dbcAMP can stimulate rabbit reproductive functions: I. Effect on ovarian folliculogenesis, ovulation and embryo production

    Directory of Open Access Journals (Sweden)

    Chrenek P.

    2012-01-01

    Full Text Available The aim of our study was to examine the influence of administration of N6,2’-dibutyryladenosine 3’5’-cyclic monophosphate (dbcAMP, a cAMP agonist, on ovarian folliculogenesis and atresia, as well as on reproductive efficiency in rabbits, whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by 20 IU/kg PMSG followed by 35 IU/kg hCG administration. Experimental animals received PMSG and hCG together with dbcAMP (at 5, 25 or 50 μg/animal. After ovulation and insemination, the animals were sacrificed. Ovaries were weighted, histological sections of ovaries were prepared, and the presence of ovulated and not ovulated follicles and different stages of atresia was evaluated by light microscopy. The eggs were flushed from the oviducts after insemination and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived zygotes and embryos reaching hatched blastocyst stage were determined. Administration of dbcAMP (at doses 25 or 50 μg/animal, but not at 5 μg/animal was able to increase the proportion of follicles with cystic and luteinization-related atresia. Furthermore, dbcAMP (50 μg/animal, but not lower doses increased the ovarian mass, number of Corpora lutea, number of harvested oocytes, zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that dbcAMP can stimulate rabbit ovarian follicle atresia, ovulation, oocyte, zygote and embryo yield and development. Furthermore, they confirm in the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of dbcAMP in improving animal reproduction and fertility.

  12. Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

    Science.gov (United States)

    Friesen, Claudia; Hormann, Inis; Roscher, Mareike; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf; Debatin, Klaus-Michael; Miltner, Erich

    2014-01-01

    Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells. PMID:24626197

  13. 产CMY型AmpC酶费劳地枸橼酸杆菌的分子学特性研究%Study on molecule characteristics of a novel CMY-type AmpC β-Lactamase from Citrobacter freundii

    Institute of Scientific and Technical Information of China (English)

    张晓坤; 徐韫健; 廖伟娇; 张东梅; 张丽梅

    2011-01-01

    目的 对产CMY型AmpC酶费劳地枸橼酸杆菌进行耐药表型及分子学特性进行研究,探讨研制新的酶抑制剂.方法 对临床分离的两株费劳地枸橼酸杆菌所产CMY型AmpC酶用纸片扩散法、三相水解试验进行耐药表型检测,以该菌总基因组DNA和质粒DNA为模板进行PCR扩增、序列分析、质粒接合试验、构建重组表达载体及AmpC酶检测.结果 两株菌株对青霉素类、头孢菌素类、喹诺酮类、氨基糖苷类抗生素均表现为耐药,对呋喃类和碳青霉烯类表现为敏感;三相水解试验结果显示该菌能水解头孢西丁;PCR扩增出大小为1 146 bp的基因片段,与GenBank上多种CMY亚型的基因序列同源性为97%;质粒接合试验证实质粒上含CMY基因,为可转移质粒.结论 两株菌株所产CMY型AmpC酶为新的CMY型头孢菌素酶,它介导了该菌对青霉素类、头孢菌素类、喹诺酮类等抗生素耐药,其耐药性能水平传播.%Objective To investigate the antibiotic phenotype and moleculelogy characteristics of novel CMY-type AmpC β-Lactamase from Citrobacter freundii,in order to triturate homologue enzyme inhibitors. Methods Slip diffusion method and three-phase hydrolyses test were used to analyze antibiotic phenotype of novel CMY-type AmpC β-Lactamase from Citrobacter freundii that was detached from clinic.The DNA and plasmid of CMY-type AmpCβ-Lactamase from Citrobacter freundii Strain was amplified by PCR、sequence analysis.Plasmid Conjugation tests、construction recombinant expression vector and AmpC induce tests. Results Two strains were resistant to penicillins , cephalosporin, quinolone, aminoglycoside , and susceptible to nitrofuran , carbapenem , The results of three-dimension test showed, AmpCβ-Lactamase from DNA Strain and recombinant strain could hydrolyz cefoxitin,The 1 l46bp DNA fragment of CMY-type AmpC β-Lactamase encoding gene sequence shared 97% amino acid identity with CMY-type AmpC β-Lactamase that

  14. Structural basis for the extended substrate spectrum of AmpC BER and structure-guided discovery of the inhibition activity of citrate against the class C β-lactamases AmpC BER and CMY-10.

    Science.gov (United States)

    Na, Jung Hyun; Cha, Sun Shin

    2016-08-01

    AmpC BER is an extended substrate spectrum class C β-lactamase with a two-amino-acid insertion in the R2 loop compared with AmpC EC2. The crystal structures of AmpC BER (S64A mutant) and AmpC EC2 were determined. Structural comparison of the two proteins revealed that the insertion increases the conformational flexibility of the R2 loop. Two citrate molecules originating from the crystallization solution were observed in the active site of the S64A mutant. One citrate molecule makes extensive interactions with active-site residues that are highly conserved among class C β-lactamases, whereas the other one is weakly bound. Based on this structural observation, it is demonstrated that citrate, a primary metabolite that is widely used as a food additive, is a competitive inhibitor of two class C β-lactamases (AmpC BER and CMY-10). Consequently, the data indicate enhancement of the flexibility of the R2 loop as an operative strategy for molecular evolution of extended-spectrum class C β-lactamases, and also suggest that the citrate scaffold is recognized by the active sites of class C β-lactamases. PMID:27487828

  15. Activation of AMP-activated protein kinase signaling pathway by adiponectin and insulin in mouse adipocytes: requirement of acyl-CoA synthetases FATP1 and Acsl1 and association with an elevation in AMP/ATP ratio.

    Science.gov (United States)

    Liu, Qingqing; Gauthier, Marie-Soleil; Sun, Lei; Ruderman, Neil; Lodish, Harvey

    2010-11-01

    Adiponectin activates AMP-activated protein kinase (AMPK) in adipocytes, but the underlying mechanism remains unclear. Here we tested the hypothesis that AMP, generated in activating fatty acids to their CoA derivatives, catalyzed by acyl-CoA synthetases, is involved in AMPK activation by adiponectin. Moreover, in adipocytes, insulin affects the subcellular localization of acyl-CoA synthetase FATP1. Thus, we also tested whether insulin activates AMPK in these cells and, if so, whether it activates through a similar mechanism. We examined these hypotheses by measuring the AMP/ATP ratio and AMPK activation on adiponectin and insulin stimulation and after knocking down acyl-CoA synthetases in adipocytes. We show that adiponectin activation of AMPK is accompanied by an ∼2-fold increase in the cellular AMP/ATP ratio. Moreover, FATP1 and Acsl1, the 2 major acyl-CoA synthetase isoforms in adipocytes, are essential for AMPK activation by adiponectin. We also show that after 40 min. insulin activated AMPK in adipocytes, which was coupled with a 5-fold increase in the cellular AMP/ATP ratio. Knockdown studies show that FATP1 and Acsl1 are required for these processes, as well as for stimulation of long-chain fatty acid uptake by adiponection and insulin. These studies demonstrate that a change in cellular energy state is associated with AMPK activation by both adiponectin and insulin, which requires the activity of FATP1 and Acsl1.

  16. Between Indian Law and Qullasuyu Nationalism. Gregorio Titiriku and the Making of AMP Indigenous Activists, 1921-1964

    Directory of Open Access Journals (Sweden)

    Waskar Ari-Chachaki

    2011-01-01

    Full Text Available In 1921, when hard-line Liberal regimes ended in Bolivia, Gregorio Titiriku, an Uru-Aymara Indian from the shores of lake Titikaka (La Paz, started 50 years of Indian intellectual activism among the Alcaldes Mayores Particulares (AMP, a 450 cell network of indigenous intellectuals. Titiriku struggled against internal colonialism and was a crucial participant in the making of AMP subaltern nationalism.  Titiriku’s ideas became a crucial part of AMP discourse, known during this time as Indian Law.  This discourse promoted the worship of Pachamama (mother earth and Achachillas (the spirit of the grandparents in the high hills of the Andes.  AMP discourse sought to rename the nation of Qullas (currently known as Aymara-Quechuas. Titiriku was especially good at creating ideas for mobilization among the AMP, such as qullasuyun wawapa (the children of the Qulla tribes in order to promote "jaqi" pride (indigenous peoples pride, and bayeta camisas (people who dress in “bayeta” in order to promote an Indian dress-code as part of a politics of identity. These ideas provide us with a privileged field for understanding of the relationship between alternative modernities and public spheres. Titiriku thus used AMP discourse to contest segregation policies and to resist mainstream civilization projects. The particularities of Indian Law and its strategic nationalism reveal the existence of alternative discourses of modernity largely forgotten in Bolivia. The analysis of AMP discourse helps us understand the longstanding presence of struggle for autonomy and hegemonic projects in Bolivia and provides us with a better comprehension of how internal colonialism and public audiences interact historically.En 1921, cuando concluyó el periodo de gobiernos liberales en Bolivia, Gregorio Titiriku, indio uru-aymara originario de las orillas del lago Titikaka (La Paz, inició cincuenta años de activismo intelectual indio entre los Alcaldes Mayores Particulares

  17. Vascular effects and cyclic AMP production produced by VIP, PHM, PHV, PACAP-27, PACAP-38, and NPY on rabbit ovarian artery

    DEFF Research Database (Denmark)

    Yao, W; Sheikh, S P; Ottesen, B;

    1996-01-01

    The relationship between vessel tone and cAMP production induced by vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), peptide histidine valine (PHV), pituitary adenylate cyclase activating polypeptide (PACAP-27 and PACAP-38), and neuropeptide Y (NPY) was investigated......-38 all increased cyclic adenosine monophosphate (cAMP) accumulation. The cAMP accumulation induced by PACAP-27 and PACAP-38 was five times higher than the cAMP content induced by the other three peptides. The peptide-induced smooth muscle relaxation did not correlate to the cAMP accumulation. NPY (10......(-7) M) markedly reversed the relaxations induced by VIP, PHM, PHV, PACAP-27, and PACAP-38, but did not influence the cAMP production induced by these peptides. In conclusion, the relaxation induced by VIP, PHM, PHV, PACAP-27, and PACAP-38 and the contraction induced by NPY are not solely related...

  18. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.

    Science.gov (United States)

    Hill, Jennifer J; Moreno, Maria J; Lam, Jean C Y; Haqqani, Arsalan S; Kelly, John F

    2009-02-01

    Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. PMID:19137551

  19. Region-selective effects of long-term lithium and carbamazepine administration on cyclic AMP levels in rat brain

    International Nuclear Information System (INIS)

    The effect of lithium and carbamazepine in the treatment of bipolar affective disorder is well established. Althougt a number of biochemical effects have been found, the exact molecular mechanisms underlying their therapeutic actions have not been elucidated nor are the target regions in the brain identified. Taken into account the important role of the cyclic AMP second messenger system in the regulation of neuronal exitability and the indications of its involvement in the pathophysiology of bipolar affective disorder, we have focused on the drug effects on cyclic AMP levels. The objectives of this investigation were to measure the effects on basal cyclic AMP levels, and to locate target regions within the rat brain after long-term administration of lithium and carbamazepine. Drug treatments were carried out for a period of 28 days. After either drug treatment the cyclic AMP level was increased 3-4 times in frontal cortex but unchanged in hippocampus, hypothalamus, thalamus, amygdala and in cerebellum. In neostratum the cyclic AMP level was decreased to about 30% after treatment with lithium. We suggest the common region-selective effect, observed for both drugs in frontal cortex, to be essential for the therapeutic actions of lithium and carbamazepine. (au)

  20. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    Science.gov (United States)

    Lobo, Miguel J; Amaral, Margarida D; Zaccolo, Manuela; Farinha, Carlos M

    2016-07-01

    Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.

  1. Management of cesium loaded AMP- Part I preparation of 137Cesium concentrate and cementation of secondary wastes

    International Nuclear Information System (INIS)

    Separation of 137cesium from High Level Waste can be achieved by use of composite-AMP, an engineered form of Ammonium Molybdo-Phosphate(AMP). Direct vitrification of cesium loaded composite AMP in borosilicate glass matrix leads to separation of water soluble molybdate phase. A proposed process describes two different routes of selective separation of molybdates and phosphate to obtain solutions of cesium concentrates. Elution of 137Cesium from composite-AMP by decomposing it under flow conditions using saturated barium hydroxide was investigated. This method leaves molybdate and phosphate embedded in the column but only 70% of total cesium loaded on column could be eluted. Alternatively composite-AMP was dissolved in sodium hydroxide and precipitation of barium molybdate-phosphate from the resultant solution, using barium nitrate was investigated by batch methods. The precipitation technique gave over 99.9% of 137Cesium activity in solutions, free of molybdates and phosphates, which is ideally suited for immobilization in borosilicate glass matrix. Detailed studies were carried out to immobilize secondary waste of 137Cesium contaminated barium molybdate-phosphate precipitates in the slag cement matrix using vermiculite and bentonite as admixtures. The cumulative fraction of 137Cs leached from the cement matrix blocks was 0.05 in 140 days while the 137Cs leach rate was 0.001 gm/cm2/d. (author)

  2. In vivo Toxicity Assessment of Antimicrobial Peptides (AMPs LR14) Derived from Lactobacillus plantarum Strain LR/14 in Drosophila melanogaster.

    Science.gov (United States)

    Gupta, Ruchi; Sarkar, Surajit; Srivastava, Sheela

    2014-03-01

    Lactic acid bacteria are known to produce antimicrobial peptides (AMPs) such as bacteriocins which can be employed to control pathogens and food spoilage microorganisms. However, their possible role as toxic agents against a eukaryotic system still remains unexplored. The present study deals with the in vivo evaluation of acute toxic effect of AMPs LR14, a mixture of AMPs isolated from Lactobacillus plantarum LR/14 on Drosophila melanogaster. The fly was used as a model system to measure the extent of toxicity of these peptides. The results showed that concentrations below 10 mg/ml are not significantly effective. When exposed to 10 mg/ml of AMPs LR14, acute toxic effect and a significant delay in the developmental cycle of the fly could be observed. Also, the weight and size of the flies were significantly reduced upon ingestion of these peptides. Higher concentrations (beyond 15 mg/ml) exerted a strong larvicidal effect. Detailed analysis on larval tissues and adult germ cells of the insect revealed deformity in cellular architecture, DNA fragmentation, and premature apoptosis, confirming that the peptides have a dose-dependent toxic property. Our studies provide the first information on the role of AMPs LR14 as an insecticidal agent.

  3. The cAMP-mediated protein kinase signal transduction pathway is involved in the pyrogenic effect of CRH in rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The primary action of corticotropin releasing hormone (CRH) is stimulation of the synthesis and release of adrenocorticotropic hormone and β-endorphin from the pituitary in response to stress. In addition, a number of studies indicate that CRH exerts other physiological actions within the central nervous system which are independent of the pituitary. These include increased body temperature and thermogenesis. However, the intracellular mechanism responsible for pyrogenic action of CRH is still unclear. The purpose of these studies was to determine whether or not cAMP was involved in the pyrogenic action of CRH in the rat. Intracerebroventricular (icv) microinjection of CRH (2.5 μg, 5.0 μg, 10 μg) caused increases in colonic temperature and hypothalamus cAMP level in conscious rats. The pyrogenic effects of CRH were abolished or markedly inhibited by prior injection (icv) of an adenylate cyclase inhibitor, 2,,3,-dideoxyadenosine (DDA, 30 μg) or an inhibitor of cAMP-dependent protein kinase, adenosine-3,,5,-(cyclic) monophosphorothionate (Rp-cAMPs, 15 μg). This is the first report demonstrating the pyrogenic effcet of centrally administration of CRH on the rat via the cAMP-mediated protein kinase signal transduction pathway.

  4. Inhibition of human cAMP-phosphodiesterase as a mechanism of the spasmolytic effect of Matricaria recutita L.

    Science.gov (United States)

    Maschi, Omar; Cero, Esther Dal; Galli, Germana V; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2008-07-01

    Mechanisms underlying the spasmolytic activity of chamomile still remain unclear. Inhibition of cAMP- and cGMP-phosphodiesterases (PDE) is one of the mechanisms operated by spasmolytic drugs. In this study, the effect of chamomile on PDE was investigated. Human platelet cAMP-PDE and recombinant PDE5A1 were assayed in the presence of infusions prepared from sifted flowers and capitula. LC-ESI-MS/MS analysis showed different compositions in infusions made with sifted flowers and capitula. Chamomile inhibited cAMP-PDE activity (IC50 = 17.9-40.5 microg/mL), while cGMP-PDE5 was less affected (-15% at 50 microg/mL). Among the individual compounds tested, only flavonoids showed an inhibitory effect (IC50 = 1.3-14.9 microM), contributing to around 39% of the infusion inhibition; other compounds responsible for cAMP-PDE inhibition still remain unknown. Although experimental evidence supporting the use of chamomile for gastrointestinal minor spasms dates back to the fifties, cAMP-PDE inhibition as a likely mechanism underlying the spasmolytic activity is reported for the first time. PMID:18553893

  5. Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California, Mexico.

    Science.gov (United States)

    Marquez-Rios, E; Pacheco-Aguilar, R; Castillo-Yañez, F J; Figueroa-Soto, C G; Ezquerra-Brauer, J M; Gollas-Galvan, T

    2008-09-01

    Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16μM/min/mg, Km of 13mM, Kcat of 3.48μM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms. PMID:26050167

  6. c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements

    Energy Technology Data Exchange (ETDEWEB)

    Inagaki, Nobuya; Seino, Yutaka; Imura, Hiroo (Kyoto Univ. (Japan)); Maekawa, Toshio; Sudo, Tatsuhiko; Ishii, Shunsuke (Inst. of Physical and Chemical Research (RIKEN), Tsukuba (Japan))

    1992-02-01

    Glucose is known to increase the cAMP concentration in pancreatic {beta} cells. To determine the mechanism by which cAMP augments insulin gene expression, the authors first identified the cAMP response elements (CREs) of human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, they constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyltransferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmic. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.

  7. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    Science.gov (United States)

    Lobo, Miguel J; Amaral, Margarida D; Zaccolo, Manuela; Farinha, Carlos M

    2016-07-01

    Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis. PMID:27206858

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

    Science.gov (United States)

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

    1997-01-01

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

  9. A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival.

    Science.gov (United States)

    Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei; Hinnebusch, Alan; Elion, Elaine A

    2003-07-15

    In mammalian cells, Ras regulates multiple effectors, including activators of mitogen-activated protein kinase (MAPK) cascades, phosphatidylinositol-3-kinase, and guanine nucleotide exchange factors (GEFs) for RalGTPases. In S. cerevisiae, Ras regulates the Kss1 MAPK cascade that promotes filamentous growth and cell integrity, but its major function is to activate adenylyl cyclase and control proliferation and survival ([; see Figure S1 in the Supplemental Data available with this article online). Previous work hints that the mating Fus3/Kss1 MAPK cascade cross-regulates the Ras/cAMP pathway during growth and mating, but direct evidence is lacking. Here, we report that Kss1 and Fus3 act upstream of the Ras/cAMP pathway to regulate survival. Loss of Fus3 increases cAMP and causes poor long-term survival and resistance to stress. These effects are dependent on Kss1 and Ras2. Activation of Kss1 by a hyperactive Ste11 MAPKKK also increases cAMP, but mating receptor/scaffold activation has little effect and may therefore insulate the MAPKs from cross-regulation. Catalytically inactive Fus3 represses cAMP by blocking accumulation of active Kss1 and by another function also shared by Kss1. The conserved RasGEF Cdc25 is a likely control point, because Kss1 and Fus3 complexes associate with and phosphorylate Cdc25. Cross-regulation of Cdc25 may be a general way that MAPKs control Ras signaling networks. PMID:12867033

  10. Simulation of hetero-junction silicon solar cells with AMPS-1D

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Como, Norberto; Morales-Acevedo, Arturo [Centro de Investigacion y de Estudios Avanzados del IPN, Electrical Engineering Department, Avenida IPN No. 2508, 07360 Mexico, D. F. (Mexico)

    2010-01-15

    Mono- and poly-crystalline silicon solar cell modules currently represent between 80% and 90% of the PV world market. The reasons are the stability, robustness and reliability of this kind of solar cells as compared to those of emerging technologies. Then, in the mid-term, silicon solar cells will continue playing an important role for their massive terrestrial application. One important approach is the development of silicon solar cells processed at low temperatures (less than 300 C) by depositing amorphous silicon layers with the purpose of passivating the silicon surface, and avoiding the degradation suffered by silicon when processed at temperatures above 800 C. This kind of solar cells is known as HIT cells (hetero-junction with an intrinsic thin amorphous layer) and are already produced commercially (Sanyo Ltd.), reaching efficiencies above 20%. In this work, HIT solar cells are simulated by means of AMPS-1D, which is a program developed at Pennsylvania State University. We shall discuss the modifications required by AMPS-1D for simulating this kind of structures since this program explicitly does not take into account interfaces with high interfacial density of states as occurs at amorphous-crystalline silicon hetero-junctions. (author)

  11. NRSF causes cAMP-sensitive suppression of sodium current in cultured hippocampal neurons

    Science.gov (United States)

    Nadeau, H.; Lester, H. A.

    2002-01-01

    The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na(+) channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1-2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na(+) channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K(+) currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic "silencer" and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

  12. Crystal structures of carbamate kinase from Giardia lamblia bound with citric acid and AMP-PNP.

    Directory of Open Access Journals (Sweden)

    Kap Lim

    Full Text Available The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP, was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123-170, which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.

  13. The effect of renal stones on serum adenosine aminohydrolase and AMP-aminohydrolase in Malaysia

    Institute of Scientific and Technical Information of China (English)

    Faridah; Yusof; Atheer; Awad; Mehde; Wesen; Adel; Mehdi; Hamid; Ghazali; Azlina; Abd; Rahman

    2015-01-01

    Objective: To verify possible associations between adenosine aminohydrolase(ADA) and AMP-aminohydrolase(AMPDA) to E3 SUMO-protein ligase NSE2(NSMCE2) in patients with renal stones. And to isolate, purify and characterize ADA in patients with renal stones and healthy group.Methods: A total of 60 renal stones patients and 50 control were enrolled in a case-control study. The blood urea, creatinine, uric acid, protein, albumin, ADA and AMPDA were measured by colorimetric tests. The serum NSMCE2 was measured by ELISA.Results: Serum ADA, AMPDA and specii c activity of enzymes showed signii cant decrease(P < 0.05) in patients with renal stones compared to control group, mean levels of sera NSMCE2 and uric acid had a signii cant increase(P < 0.01 and P < 0.05, respectively) in patients compared to control group.Conclusions: The present study suggests that ADA, AMP deaminase and NSMCE2 can be used as a indicator to monitor the DNA damage and inl ammation disorders in the patients with kidney stones.

  14. Isolation of 137-Cs in tuna fish by ammonium phosphomolybdate (AMP)

    International Nuclear Information System (INIS)

    The isolation of 137-Cs in tuna fish (Neithunnus macroptenus) has been carried out using AMP. The energy of 137-Cs was measure using NaI(T1) detector connected to a Multi Channel Analyzer (MCA). In standard solution of 137-Cs with activity 160.74 and 80.37 pCi, the addition of 120 mg AMP and 7 hours of absorption time, caused 97% of 137-Cs in the solution was absorbed. The destruction of tuna fish was carried out using two methods namely by wet destruction with sulfuric acid, nitrit acid and hydrogen peroxid mixture and by wet destruction with hydrogen peroxide. The applications of this technique of tuna fish was carried out by spiking 137-Cs standard into the fish before being destructed. The recovery obtained from wet destruction with H2O2 was about 80%, while from destruction with acid mixtures and H2O2 was only about 47%. (authors). 3 refs, 3 tabs

  15. Soybean diet improves insulin secretion through activation of cAMP/PKA pathway in rats.

    Science.gov (United States)

    Veloso, Roberto V; Latorraca, Márcia Q; Arantes, Vanessa C; Reis, Marise A B; Ferreira, Fabiano; Boschero, Antonio C; Carneiro, Everardo M

    2008-11-01

    Maternal malnutrition leads to permanent alterations in insulin secretion of offspring and the soybean diet contributes to improve insulin release. At least a soy component, genistein, seems to increase the insulin secretion by activating the cAMP/PKA and PLC/PKC pathways. Here, we investigated the effect of the soybean diet on the expression of PKAalpha and PKCalpha, and insulin secretion in response to glucose and activators of adenylate cyclase and PKC in adult pancreatic rat islets. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet until 90 days of life. The soybean diet improved the insulin response to a physiological concentration of glucose in control islets, but only in the presence of supra-physiological concentrations of glucose in islets from CR and SR groups. PMA also improved the insulin response in islets of SC and SR groups. The expression of PKCalpha was similar in all groups. Forskolin increased the insulin secretion; however, the magnitude of the increment was lower in islets from CR and SR groups than in control animals and in those from rats maintained with soybean diet than in rats fed with casein diet. The PKAalpha expression was similar between SR and CR groups and lower in SC than in CC islets. Thus, soybean diet improved the secretory pattern of beta cells, at least in part, by activating the cAMP/PKA-signaling cascade. PMID:18430554

  16. Solving the Monge-Amp\\`ere Equations for the Inverse Reflector Problem

    CERN Document Server

    Brix, Kolja; Platen, Andreas

    2014-01-01

    The inverse reflector problem arises in geometrical nonimaging optics: Given a light source and a target, the question is how to design a reflecting free-form surface such that a desired light density distribution is generated on the target, e.g., a projected image on a screen. This optical problem can mathematically be understood as a problem of optimal transport and equivalently be expressed by a secondary boundary value problem of the Monge-Amp\\`ere equation, which consists of a highly nonlinear partial differential equation of second order and constraints. In our approach the Monge-Amp\\`ere equation is numerically solved using a collocation method based on tensor-product B-splines, in which nested iteration techniques are applied to ensure the convergence of the nonlinear solver and to speed up the calculation. In the numerical method special care has to be taken for the constraint: It enters the discrete problem formulation via a Picard-type iteration. Numerical results are presented as well for benchmar...

  17. Role of AMP-activated protein kinase in metabolic depression in animals.

    Science.gov (United States)

    Rider, Mark H

    2016-01-01

    AMP-activated protein kinase (AMPK) is a highly conserved eukaryotic protein serine/threonine kinase that controls cellular and whole body energy homoeostasis. AMPK is activated during energy stress by a rise in AMP:ATP ratio and maintains energy balance by phosphorylating targets to switch on catabolic ATP-generating pathways, while at the same time switching off anabolic ATP-consuming processes. Metabolic depression is a strategy used by many animals to survive environmental stress and has been extensively studied across phylogeny by comparative biochemists and physiologists, but the role of AMPK has only recently been addressed. This review first deals with the evolution of AMPK in eukaryotes (excluding plants and fungi) and its regulation. Changes in adenine nucleotides and AMPK activation are described in animals during environmental energy stress, before considering the involvement of AMPK in controlling β-oxidation, fatty acid synthesis, triacylglycerol mobilization and protein synthesis. Lastly, strategies are presented to validate the role of AMPK in mediating metabolic depression by phosphorylating downstream targets.

  18. Polyphosphate, cyclic AMP, guanosine tetraphosphate, and c-di-GMP reduce in vitro Lon activity.

    Science.gov (United States)

    Osbourne, Devon O; Soo, Valerie W C; Konieczny, Igor; Wood, Thomas K

    2014-01-01

    Lon protease is conserved from bacteria to humans and regulates cellular processes by degrading different classes of proteins including antitoxins, transcriptional activators, unfolded proteins, and free ribosomal proteins. Since we found that Lon has several putative cyclic diguanylate (c-di-GMP) binding sites and since Lon binds polyphosphate (polyP) and lipid polysaccharide, we hypothesized that Lon has an affinity for phosphate-based molecules that might regulate its activity. Hence we tested the effect of polyP, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), guanosine tetraphosphate (ppGpp), c-di-GMP, and GMP on the ability of Lon to degrade α-casein. Inhibition of in vitro Lon activity occurred for polyP, cAMP, ppGpp, and c-di-GMP. We also demonstrated by HPLC that Lon is able to bind c-di-GMP. Therefore, four cell signals were found to regulate the activity of Lon protease.

  19. Cytoplasmic cAMP-sensing domain of hyperpolarization-activated cation (HCN) channels uses two structurally distinct mechanisms to regulate voltage gating

    OpenAIRE

    Wicks, Nadine L.; Wong, Tammy; Sun, Jinyi; Madden, Zarina; Young, Edgar C.

    2010-01-01

    Voltage gating of hyperpolarization-activated cation (HCN) channels is potentiated by direct binding of cAMP to a cytoplasmic cAMP-sensing domain (CSD). When unliganded, the CSD inhibits hyperpolarization-dependent opening of the HCN channel gate; cAMP binding relieves this autoinhibition so that opening becomes more favorable thermodynamically. This autoinhibition-relief mechanism is conserved with that of several other cyclic nucleotide receptors using the same ligand-binding fold. Besides ...

  20. Phosphodiesterase 4 inhibitors and db-cAMP inhibit TNF-α release from human mononuclear cells. Effects of cAMP and cGMP-dependent protein kinase inhibitors

    Directory of Open Access Journals (Sweden)

    A. Hichami

    1996-01-01

    Full Text Available We investigated the effects of specific inhibitors of cAMP-dependent protein kinase (PKA and cGMP-dependent protein kinase (PKG on the inhibitory activity of phosphodiesterase (PDE type 4 inhibitors and of the cell permeable analogue of cAMP, db-cAMP on LPS-induced TNF-α release from human mononuclear cells. Incubation from 30 min of mononuclear cells with dbcAMP (10−5 to 10−3 M, rolipram (10−9 M to 10−5 M or Ro 20-1724 (10−9 M to 10−5 M significantly inhibited LPS-induced TNF-α release. When mononuclear cells were preincubated for 30 min with the selective PKA inhibitor, H89 (10−4 M, but not with the selective PKG inhibitor, Rp-8-pCPT-cGMPs (10−4 M, a significant reduction of the inhibitory effect of db-cAMP was noted. Thirty min incubation of mononuclear cells with Rp-8-pCPT-cGMPs induced a significant reduction of the inhibitory activities of both rolipram and Ro 20-1724 (10−9 to 10−5 M on LPS-induced TNF-α release, whereas H89 elicited a moderate, but significant inhibition. The present data indicate that db-cAMP inhibits TNF-α release from human mononuclear cells through a PKA-dependent mechanism. In contrast, PDE 4 inhibitors elicit their in vitro anti-inflammatory activities via a PKG-dependent rather than PKA-dependent activation.

  1. The adhesion modulation protein, AmpA localizes to an endocytic compartment and influences substrate adhesion, actin polymerization and endocytosis in vegetative Dictyostelium cells

    Directory of Open Access Journals (Sweden)

    Noratel Elizabeth F

    2012-11-01

    Full Text Available Abstract Background AmpA is a secreted 24Kd protein that has pleiotropic effects on Dictyostelium development. Null mutants delay development at the mound stage with cells adhering too tightly to the substrate. Prestalk cells initially specify as prespore cells and are delayed in their migration to the mound apex. Extracellular AmpA can rescue these defects, but AmpA is also necessary in a cell autonomous manner for anterior like cells (ALCs to migrate to the upper cup. The ALCs are only 10% of the developing cell population making it difficult to study the cell autonomous effect of AmpA on the migration of these cells. AmpA is also expressed in growing cells, but, while it contains a hydrophobic leader sequence that is cleaved, it is not secreted from growing cells. This makes growing cells an attractive system for studying the cell autonomous function of AmpA. Results In growing cells AmpA plays an environment dependent role in cell migration. Excess AmpA facilitates migration on soft, adhesive surfaces but hinders migration on less adhesive surfaces. AmpA also effects the level of actin polymerization. Knockout cells polymerize less actin while over expressing cells polymerize more actin than wild type. Overexpression of AmpA also causes an increase in endocytosis that is traced to repeated formation of multiple endocytic cups at the same site on the membrane. Immunofluorescence analysis shows that AmpA is found in the Golgi and colocalizes with calnexin and the slow endosomal recycling compartment marker, p25, in a perinuclear compartment. AmpA is found on the cell periphery and is endocytically recycled to the perinuclear compartment. Conclusion AmpA is processed through the secretory pathway and traffics to the cell periphery where it is endocytosed and localizes to what has been defined as a slow endosomal recycling compartment. AmpA plays a role in actin polymerization and cell substrate adhesion. Additionally AmpA influences cell

  2. Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL among AmpC-producing Enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    R M Shoorashetty

    2011-01-01

    Full Text Available Purpose: Extended spectrum β-lactamase (ESBL and AmpC β-lactamase are important mechanisms of betalactam resistance among Enterobacteriaceae . The ESBL confirmation test described by Clinical Laboratory Standards Institute (CLSI is in routine use. This method fails to detect ESBL in the presence of AmpC. Therefore, we compared two different ESBL detection methods against the CLSI confirmatory test. Materials and Methods: A total 200 consecutive clinical isolates of Enterobacteriaceae from various clinical samples were tested for ESBL production using (i CLSI described phenotypic confirmatory test (PCT, (ii boronic acid disk potentiation test and (iii cefepime-CA disk potentiation method. AmpC confirmation was done by a modified three-dimensional test. Results: Among total 200 Enterobacteriaceae isolates, 82 were only ESBL producers, 12 were only AmpC producers, 55 were combined ESBL and AmpC producers, 14 were inducible AmpC producers and 37 isolates did not harboured any enzymes. The CLSI described PCT detected ESBL-producing organisms correctly but failed to detect 36.3% of ESBLs among combined enzyme producers. The boronic acid disk potentiation test reliably detected all ESBL, AmpC, and combined enzyme producers correctly. The cefepime-CA method detected all ESBLs correctly but another method of AmpC detection has to be adopted. Conclusion: The use of boronic acid in disk diffusion testing along with the CLSI described PCT enhances ESBL detection in the presence of AmpC betalactamases.

  3. Calcium-linked increase in coupled cAMP synthesis and hydrolysis is an early event in cholinergic and. beta. -adrenergic stimulation of parotid secretion

    Energy Technology Data Exchange (ETDEWEB)

    Deeg, M.A.; Graeff, R.M.; Walseth, T.F.; Goldberg, N.D. (Univ. of Minnesota, Minneapolis (USA))

    1988-11-01

    The dynamics and compartmental characteristics of cAMP metabolism were examined by {sup 18}O labeling of cellular adenine nucleotide {alpha} phosphoryls in rat parotid gland stimulated to secrete with {beta}-adrenergic and cholinergic agents. The secretory response occurred in association with a rapidly increased rate of cAMP hydrolysis apparently coordinated with an equivalent increase in the rate of cAMP synthesis, since the cellular concentration of cAMP remained unchanged. The magnitude of this metabolic response was equivalent to the metabolism of 10-75 times the cellular content of cAMP within the first minute of stimulation. This increased metabolic rate occurred only during the early (1-3 min) period of stimulation, in what appeared to be an exclusive cellular compartment distinguished by a unique distribution of {sup 18}O among adenine nucleotide {alpha} phosphoryls. This {sup 18}O distribution contrasted with that produced by forskolin, which increased cellular cAMP concentration and elicited only a delayed response missing the early secretory component. The early acceleration of cAMP metabolism appeared linked to a stimulus-induced increase in intracellular Ca{sup 2+} concentration, since the Ca{sup 2+} ionophore ionomycin produced the same metabolic response in association with secretion. These observations suggest that cAMP metabolism is involved in stimulus-secretion coupling by a Ca{sup 2+}-linked mechanism different from that in which cAMP plays the role of a second messenger.

  4. cAMP dependent and independent regulation of thyroglobulin synthesis by two clones of the OVNIS 6H thyroid cell line.

    Science.gov (United States)

    Aouani, A; Hovsépian, S; Fayet, G

    1987-07-01

    The hormonal regulation of thyroglobulin synthesis has been studied using two independent clones of the OVNIS 6H cell line. Insulin, hydrocortisone and TSH were able to stimulate thyroglobulin synthesis, whereas transferrin, somatostatin and glycyl-histidyl-lysine were without effect. Insulin stimulated thyroglobulin synthesis without affecting cAMP production. Hydrocortisone, when combined with insulin was a stimulator too; this stimulation was not accompanied by an increase in cAMP. TSH alone was unable to stimulate either cAMP or thyroglobulin synthesis. The stimulatory effect of TSH on thyroglobulin synthesis took place only when combined with insulin or insulin plus hydrocortisone, and was mediated by cAMP. Consequently, insulin and hydrocortisone stimulated thyroglobulin synthesis by cAMP-independent mechanisms, whereas TSH acted via the cAMP system. Forskolin mimicked TSH effects on cAMP and thyroglobulin synthesis. Calf serum inhibited cAMP and thyroglobulin production. Optimal cAMP and thyroglobulin synthesis as well as TSH responsiveness were obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH. PMID:3040495

  5. The relationship between some beta-adrenergic mediated responses and plasma concentrations of adrenaline and cyclic AMP in man

    DEFF Research Database (Denmark)

    Philipsen, E K; Myhre, John Gabriel; Larsen, S;

    1990-01-01

    concentrations at low adrenaline infusion rates was prevented, whereas a small increase in cyclic AMP was found at high adrenaline infusion rates, probably owing to incomplete beta-receptor blockade. Likewise, the adrenaline-induced increments in blood substrates (glucose, lactate, glycerol and beta......To test the hypothesis that increments in plasma cyclic AMP during beta-adrenergic stimulation reflect integrated second messenger function of the tissues activated by the agonist, graded adrenaline infusion resulting in plasma adrenaline concentrations within the physiological range was performed...... hydroxybutyric acid) were significantly reduced but not completely prevented by beta-blockade. We conclude that an altered relationship between beta-agonist concentrations and plasma cyclic AMP may provide evidence for the existence of differences in beta-adrenergic sensitivity in man....

  6. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner.

    Science.gov (United States)

    Kottyan, Leah C; Collier, Ann R; Cao, Khanh H; Niese, Kathryn A; Hedgebeth, Megan; Radu, Caius G; Witte, Owen N; Khurana Hershey, Gurjit K; Rothenberg, Marc E; Zimmermann, Nives

    2009-09-24

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5'-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase-dependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65(-/-) mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP- and GPR65-dependent manner. PMID:19641187

  7. Antimicrobial peptide (Cn-AMP2) from liquid endosperm of Cocos nucifera forms amyloid-like fibrillar structure.

    Science.gov (United States)

    Gour, Shalini; Kaushik, Vibha; Kumar, Vijay; Bhat, Priyanka; Yadav, Subhash C; Yadav, Jay K

    2016-04-01

    Cn-AMP2 is an antimicrobial peptide derived from liquid endosperm of coconut (Cocos nucifera). It consists of 11 amino acid residues and predicted to have high propensity for β-sheet formation that disposes this peptide to be amyloidogenic. In the present study, we have examined the amyloidogenic propensities of Cn-AMP2 in silico and then tested the predictions under in vitro conditions. The in silico study revealed that the peptide possesses high amyloidogenic propensity comparable with Aβ. Upon solubilisation and agitation in aqueous buffer, Cn-AMP2 forms visible aggregates that display bathochromic shift in the Congo red absorbance spectra, strong increase in thioflavin T fluorescence and fibrillar morphology under transmission electron microscopy. All these properties are typical of an amyloid fibril derived from various proteins/peptides including Aβ. PMID:27028204

  8. Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model

    International Nuclear Information System (INIS)

    Multiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as a target for inhibiting the growth of multiple myeloma cells. As a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice. In contrast to the effect on Reh cells, 50 μM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo. Elevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma

  9. Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics.

    Science.gov (United States)

    Golkowski, Martin; Shimizu-Albergine, Masami; Suh, Hyong Won; Beavo, Joseph A; Ong, Shao-En

    2016-07-01

    Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion

  10. Performance of ACMG-AMP Variant-Interpretation Guidelines among Nine Laboratories in the Clinical Sequencing Exploratory Research Consortium.

    Science.gov (United States)

    Amendola, Laura M; Jarvik, Gail P; Leo, Michael C; McLaughlin, Heather M; Akkari, Yassmine; Amaral, Michelle D; Berg, Jonathan S; Biswas, Sawona; Bowling, Kevin M; Conlin, Laura K; Cooper, Greg M; Dorschner, Michael O; Dulik, Matthew C; Ghazani, Arezou A; Ghosh, Rajarshi; Green, Robert C; Hart, Ragan; Horton, Carrie; Johnston, Jennifer J; Lebo, Matthew S; Milosavljevic, Aleksandar; Ou, Jeffrey; Pak, Christine M; Patel, Ronak Y; Punj, Sumit; Richards, Carolyn Sue; Salama, Joseph; Strande, Natasha T; Yang, Yaping; Plon, Sharon E; Biesecker, Leslie G; Rehm, Heidi L

    2016-06-01

    Evaluating the pathogenicity of a variant is challenging given the plethora of types of genetic evidence that laboratories consider. Deciding how to weigh each type of evidence is difficult, and standards have been needed. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published guidelines for the assessment of variants in genes associated with Mendelian diseases. Nine molecular diagnostic laboratories involved in the Clinical Sequencing Exploratory Research (CSER) consortium piloted these guidelines on 99 variants spanning all categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign). Nine variants were distributed to all laboratories, and the remaining 90 were evaluated by three laboratories. The laboratories classified each variant by using both the laboratory's own method and the ACMG-AMP criteria. The agreement between the two methods used within laboratories was high (K-alpha = 0.91) with 79% concordance. However, there was only 34% concordance for either classification system across laboratories. After consensus discussions and detailed review of the ACMG-AMP criteria, concordance increased to 71%. Causes of initial discordance in ACMG-AMP classifications were identified, and recommendations on clarification and increased specification of the ACMG-AMP criteria were made. In summary, although an initial pilot of the ACMG-AMP guidelines did not lead to increased concordance in variant interpretation, comparing variant interpretations to identify differences and having a common framework to facilitate resolution of those differences were beneficial for improving agreement, allowing iterative movement toward increased reporting consistency for variants in genes associated with monogenic disease.

  11. [C-AMP concentration in various organs of female rats and in human ovaries with aging (author's transl)].

    Science.gov (United States)

    Arima, M

    1982-02-01

    The change in 3',5'-cyclic adenosine monophosphate (c-AMP) concentration was observed in various organs of rats in gonadal cycle in adult group and with aging (30, 70, 100, 120 weeks), and in human ovaries with aging. 1) The average c-AMP concentration of ovaries of rats showed a significant change with estrus cycle and was higher in the following sequence: proestrus, diestrus II, diestrus I and estrus phase. This tendency was also seen in hypothalamus and pituitary, but was not statistically significant, 2) The average c-AMP concentration in tissues began to decline significantly from 70 weeks in cerebral cortex and hypothalamus, and from 80 weeks in ovaries. However, on the other hand the concentrations in pituitary, liver and adrenal declined markedly from 100 weeks. 3) The c-AMP in ovaries of 80 weeks rats by pregnant mare serum (PMS) road increased by 0.5-fold in concentration, and by 0.6-fold in whole tissue relative to that of 30 weeks rats. 4) A significant difference in serum LH and FSH level between ovarian artery and vein was not found in cycling mature group, non-cycling climacteric group and post-menopausal group of women. 5) Both average concentrations and total values of c-AMP in ovaries of non-cycling climacteric and post-menopausal women were lower than those of mature cycling women. This fact may imply a different response by ovarian tissues such as corpus luteum, follicle and other tissues to gonadotropin. From these results of c-AMP in tissues, it is concluded that the decline of ovarian function with aging of rats was relatively earlier than pituitary, although being delayed compared with hypothalamus, and were the ovarian function in humans declined in the premenopausal period.

  12. A new cAMP response element in the transcribed region of the human c-fos gene.

    OpenAIRE

    Härtig, E; Loncarević, I F; Büscher, M.; Herrlich, P; Rahmsdorf, H J

    1991-01-01

    In NIH 3T3 cells the c-fos gene is induced rapidly and transiently by cAMP. As shown by the analysis of 3T3 cells stably transfected with promoter mutants of the human c-fos gene this induction does not depend on the dyad symmetry element (position -320 to -300), but involves at least two other non-related sites: an element located around position -60 resembling the cAMP response element of the fibronectin and somatostatin genes (which has been described before), and an element located betwee...

  13. Nuevas dianas de actuación de la proteína quinasa activada por AMP (AMPK)

    OpenAIRE

    Solaz Fuster, María del Carmen

    2007-01-01

    RESUMEN En múltiples tejidos de mamíferos, la proteína quinasa activada por AMP (AMPK) controla el metabolismo de la glucosa y de los lípidos. Esta importante función de AMPK como sensor energético conservado evolutivamente y regulador clave del metabolismo estaría, además, apoyada por el papel de su ortólogo en el metabolismo de la glucosa del eucariota unicelular Saccharomyces cerevisiae. La proteína se activa en respuesta a un aumento en la relación de AMP respecto a ATP en el interior ...

  14. Inhibition of human platelet aggregation by dihydropyrano- and dihydrofuranocoumarins, a new class of cAMP-phosphodiesterase inhibitors

    DEFF Research Database (Denmark)

    Thastrup, Ole; Knudsen, J B; Lemmich, J;

    1985-01-01

    Certain esters of dihydropyranocoumarin and dihydrofuranocoumarin alcohols have previously been shown to inhibit the cAMP-phosphodiesterase from bovine heart. We now report that these naturally occurring coumarins inhibit the high affinity (Km = 1.1 microM) cAMP-phosphodiesterase from human...... platelets with activities that closely correlate with those obtained using phosphodiesterase from bovine heart tissue. Additionally the coumarins inhibit the aggregation of human platelets induced with ADP, adrenaline and collagen with activities comparable to those of dipyridamole. A lack of significant...

  15. Blocking of Exchange Proteins Directly Activated by cAMP Leads to Reduced Replication of Middle East Respiratory Syndrome Coronavirus

    Science.gov (United States)

    Tao, Xinrong; Mei, Feng; Agrawal, Anurodh; Peters, Clarence J.; Ksiazek, Thomas G.

    2014-01-01

    The outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections and diseases represents a potential threat for worldwide spread and requires development of effective therapeutic strategies. In this study, we revealed a novel positive function of an exchange protein directly activated by cyclic AMP 1 (cAMP-1; Epac-1) on MERS-CoV replication. Specifically, we have shown that Epac-specific inhibitor treatment or silencing Epac-1 gene expression rendered cells resistant to viral infection. We believe Epac-1 inhibitors deserve further study as potential therapeutic agents for MERS-CoV infection. PMID:24453361

  16. First Results of AMP surveys for large areas (>100ha) with decimeter resolutions in Archaeology

    Science.gov (United States)

    Dabas, Michel

    2010-05-01

    The work presented here takes place within the BREBEMI project which is a motorway project between the town of Milano and Brescia in Italy. For the first time, a set of non-invasive tools is used systematically in order to prevent the archaeological risk before the construction of a motorway. This innovative project relies both on: - the systematic use of a GIS (Geographical Information System) for integration of all data at every stage of the project (from data acquisition in the field to interpretation and field check), - the use of new continuous high productivity geophysical techniques able to survey several hectares a day with a resolution of a few decimetres (resistivity:ARP(c) and magnetics: AMP), and aerial LIDAR data (acquisition of Digital Elevation Model). - the integration of all different sources of information (geophysics, aerial photos, documentation, historical evidences but also field truething of anomalies) For the first time all these techniques are not only used at the level of the site, but also at the level of the landscape. The objectives of this project is to reduce to a maximum the uncertainty linked to the presence of archaeological remains (in particular delimitation of areas not to be excavated due to both positive and/or negative archaeological artefacts). In the presentation we will focus on geophysics. We have combined the use of continuous electrical imaging techniques (ARP(c)) and continuous magnetic imaging techniques(AMP, Geocarta, Paris). The first method enables measurement of apparent electrical resistivity for 3 depths of investigation at the same time. The AMP method enables the measurement of magnetic vertical gradient of the Earth Magnetic Field. All data are acquired with Quad bykes-towed instruments controlled in real-time with a GIS software: sampling mesh is of the order of a few centimetres in-line and 50cm across lines. No external topography is done and all profiles are computed by the GIS and a differential GPS for

  17. 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

  18. AMP kinase expression and activity in human skeletal muscle: effects of immobilization, retraining, and creatine supplementation

    DEFF Research Database (Denmark)

    Eijnde, Bert O.; Derave, Wim; Wojtaszewski, Jørgen;

    2005-01-01

    The effects of leg immobilization and retraining in combination with oral creatine intake on muscle AMP-activated protein kinase (AMPK) protein expression and phosphorylation status were investigated. A double-blind trial was performed in young healthy volunteers (n = 22). A cast immobilized the...... right leg for 2 wk, whereafter the knee-extensor muscles of that leg were retrained for 6 wk. Half of the subjects received creatine monohydrate throughout the study (Cr; from 15 g down to 2.5 g daily), and the others ingested placebo (P; maltodextrin). Before and after immobilization and retraining...... immobilization-induced muscle inactivity for 2 wk does not alter AMPK a1-, a2-, and ß2-subunit expression or a-AMPK phosphorylation status. Furthermore, the present observations indicate that AMPK probably is not implicated in the previously reported beneficial effects of oral creatine supplementation on muscle...

  19. Pharmacological Targeting of AMP-Activated Protein Kinase and Opportunities for Computer-Aided Drug Design.

    Science.gov (United States)

    Miglianico, Marie; Nicolaes, Gerry A F; Neumann, Dietbert

    2016-04-14

    As a central regulator of metabolism, the AMP-activated protein kinase (AMPK) is an established therapeutic target for metabolic diseases. Beyond the metabolic area, the number of medical fields that involve AMPK grows continuously, expanding the potential applications for AMPK modulators. Even though indirect AMPK activators are used in the clinics for their beneficial metabolic outcome, the few described direct agonists all failed to reach the market to date, which leaves options open for novel targeting methods. As AMPK is not actually a single molecule and has different roles depending on its isoform composition, the opportunity for isoform-specific targeting has notably come forward, but the currently available modulators fall short of expectations. In this review, we argue that with the amount of available structural and ligand data, computer-based drug design offers a number of opportunities to undertake novel and isoform-specific targeting of AMPK. PMID:26510622

  20. Distribution and function of 3',5'-Cyclic-AMP phosphodiesterases in the human ovary

    DEFF Research Database (Denmark)

    Petersen, T S; Kristensen, S G; Jeppesen, J V;

    2015-01-01

    The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary....... Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found...... that PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A was not detected. PDE8B was differentially expressed during folliculogenesis. In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone secretion while PDE4 inhibition increased forskolin-stimulated progesterone...

  1. Designing Illumination Lenses and Mirrors by the Numerical Solution of Monge-Amp\\`ere Equations

    CERN Document Server

    Brix, Kolja; Platen, Andreas

    2015-01-01

    We consider the inverse refractor and the inverse reflector problem. The task is to design a free-form lens or a free-form mirror that, when illuminated by a point light source, produces a given illumination pattern on a target. Both problems can be modeled by strongly nonlinear second-order partial differential equations of Monge-Amp\\`ere type. In [Math. Models Methods Appl. Sci. 25 (2015), pp. 803--837, DOI: 10.1142/S0218202515500190] the authors have proposed a B-spline collocation method which has been applied to the inverse reflector problem. Now this approach is extended to the inverse refractor problem. We explain in depth the collocation method and how to handle boundary conditions and constraints. The paper concludes with numerical results of refracting and reflecting optical surfaces and their verification via ray tracing.

  2. Simulation of CIGS Thin Film Solar Cells Using AMPS-1D

    Directory of Open Access Journals (Sweden)

    J.R. Ray

    2011-01-01

    Full Text Available The solar cell structure based on copper indium gallium diselenide (CIGS as the absorber layer, cadmium sulfide (CdS as a buffer layer un-doped (i and Aluminium (Al doped zinc oxide (ZnO as a window layer was simulated using the one dimensional simulation program called analysis of microelectronic and photonic structures (AMPS-1D. In the simulation, the thickness of CIGS layer was varied from 300 to 3000 nm. The rest of layer’s thicknesses were kept constant, viz. 60 nm for CdS, and 80 nm and 500 nm for i- and Al-ZnO, respectively. By varying thickness of CIGS layer the simulated device performance was demonstrate in the form of current-voltage (I-V characteristics and quantum efficiency (QE.

  3. Preparation of Inverse Microemulsion of Water Soluble P(AM-co-AMPS)%水溶性P(AM-co-AMPS)反相微乳胶的制备研究

    Institute of Scientific and Technical Information of China (English)

    聂莹; 晁芬

    2006-01-01

    根据绘制的AM/AMPS/H2O-Kerosene-Span80/Tween80体系的拟三元相图,配制了系列AM/AMPS反相微乳液,考察了相关因素对AM/AMPS反相微乳液聚合的影响.发现当混合单体中m(AM):m(AMPS)=5时,所得P(AM-co-AMPS)的相对分子质量最大,且随单体质量分数的增加而增大,随反应温度的升高和乳化剂质量分数的增加而减小,和随引发剂过硫酸铵(APS)质量分数、体系pH值的增大呈现出先增大后减小的变化趋势.在适宜条件下,制备了固含量为31%,相对平均分子质量为9.8×106,稳定、透明的P(AM-co-AMPS)反相微乳胶.

  4. Glucose starvation-induced dispersal of Pseudomonas aeruginosa biofilms is cAMP and energy dependent.

    Directory of Open Access Journals (Sweden)

    Tran T Huynh

    Full Text Available Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ. In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA. In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival.

  5. CFTR: a hub for kinases and crosstalk of cAMP and Ca2+.

    Science.gov (United States)

    Kunzelmann, Karl; Mehta, Anil

    2013-09-01

    Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). The resulting disease is pleiotropic consistent with the idea that CFTR acts as a node within a network of signalling proteins. CFTR is not only a regulator of multiple transport proteins and controlled by numerous kinases but also participates in many signalling pathways that are disrupted after expression of its commonest mutant (F508del-CFTR). It operates in membrane compartments creating a scaffold for cytoskeletal elements, surface receptors, kinases and phosphodiesterases. CFTR is exposed to membrane-local second messengers such that a CFTR-interacting, low cellular energy sensor kinase (AMP- and ADP-activated kinase, AMPK) signals through a high energy phosphohistidine protein kinase (nucleoside diphosphate kinase, NDPK). CFTR also translocates a Ca(2+)-dependent adenylate cyclase to its proximity so that a rigid separation between cAMP-dependent and Ca(2+)-dependent regulation of Cl(-) transport becomes obsolete. In the presence of wild-type CFTR, parallel activation of CFTR and outwardly rectifying anoctamin 6 Cl(-) channels is observed, while the Ca(2+)-activated anoctamin 1 Cl(-) channel is inhibited. In contrast, in CF cells, CFTR is missing/mislocalized and the outwardly rectifying chloride channel is attenuated while Ca(2+)-dependent Cl(-) secretion (anoctamin 1) appears upregulated. Additionally, we consider the idea that F508del-CFTR when trapped in the endoplasmic reticulum augments IP3-mediated Ca(2+) release by providing a shunt pathway for Cl(-). CFTR and the IP3 receptor share the characteristic that they both assemble their partner proteins to increase the plasticity of their hub responses. In CF, the CFTR hub fails to form at the plasma membrane, with widespread detrimental consequences for cell signalling.

  6. Poly(NIPAm-AMPS) nanoparticles for targeted delivery of anti-inflammatory cell penetrating peptides

    Science.gov (United States)

    Bartlett, Rush Lloyd, II

    Inflammatory diseases such as osteoarthritis and rheumatoid arthritis cause $127.8 billion in US healthcare expenditures each year and are the cause of disability for 27% of disabled persons in the United States. Current treatment options rarely halt disease progression and often result in significant unwanted and debilitating side effects. Our laboratory has previously developed a family of cell penetrating peptides (CPPs) which inhibit the activity of mitogen activated protein kinase activate protein kinase 2 (MK2). MK2 mediates the inflammatory response by activating Tristetraprline (TTP). Once activated, TTP rapidly stabilizes AU rich regions of pro-inflammatory cytokine mRNA which allows translation of pro-inflammatory cytokines to occur. Blocking MK2 with our labs CPPs yields a decrease in inflammatory activity but CPPs by are highly non specific and prone to rapid enzymatic degradation in vivo.. In order to increase the potency of MK2 inhibiting CPPs we have developed a novel nanoparticle drug carrier composed of poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid). This drug carrier has been shown to have preliminary efficacy in vitro and ex vivo for suppressing pro-inflammatory cytokine production when releasing CPPs. This thesis will present progress made on three aims: Specific Aim 1) Create and validate a NIPAm based drug delivery system that mimics the binding and release previously observed between cell penetrating peptides and glycosaminoglycans. Specific Aim 2) Engineer degradability into poly(NIPAm-AMPS) nanoparticles to enable more drug to be released and qualify that system in vitro. Specific Aim 3) Validate poly(NIPAm-AMPS) nanoparticles for targeted drug delivery in an ex vivo inflammatory model. Overall we have developed a novel anionic nanoparticle system that is biocompatible and efficient at loading and releasing cell penetrating peptides to inflamed tissue. Once loaded with a CPP the nanoparticle drug complex is

  7. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    Science.gov (United States)

    Breit, Andreas; Wicht, Kristina; Boekhoff, Ingrid; Glas, Evi; Lauffer, Lisa; Mückter, Harald; Gudermann, Thomas

    2016-07-01

    Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction. PMID:27144291

  8. The Deacylation Mechanism of AmpC [beta]-Lactamase at Ultrahigh Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu; Minasov, George; Roth, Tomer A.; Prati, Fabio; Shoichet, Brian K. (Degli); (NWU); (UCSF)

    2010-03-05

    {beta}-Lactamases confer bacterial resistance to {beta}-lactam antibiotics, such as penicillins. The characteristic class C {beta}-lactamase AmpC catalyzes the reaction with several key residues including Ser64, Tyr150, and Lys67. Here, we describe a 1.07 {angstrom} X-ray crystallographic structure of AmpC {beta}-lactamase in complex with a boronic acid deacylation transition-state analogue. The high quality of the electron density map allows the determination of many proton positions. The proton on the Tyr150 hydroxyl group is clearly visible and is donated to the boronic oxygen mimicking the deacylation water. Meanwhile, Lys67 hydrogen bonds with Ser64O{gamma}, Asn152O{delta}1, and the backbone oxygen of Ala220. This suggests that this residue is positively charged and has relinquished the hydrogen bond with Tyr150 observed in acyl-enzyme complex structures. Together with previous biochemical and NMR studies, these observations indicate that Tyr150 is protonated throughout the reaction coordinate, disfavoring mechanisms that involve a stable tyrosinate as the general base for deacylation. Rather, the hydroxyl of Tyr150 appears to be well positioned to electrostatically stabilize the negative charge buildup in the tetrahedral high-energy intermediate. This structure, in itself, appears consistent with a mechanism involving either Tyr150 acting as a transient catalytic base in conjunction with a neutral Lys67 or the lactam nitrogen as the general base. Whereas mutagenesis studies suggest that Lys67 may be replaced by an arginine, disfavoring the conjugate base mechanism, distinguishing between these two hypotheses may ultimately depend on direct determination of the pKa of Lys67 along the reaction coordinate.

  9. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    Science.gov (United States)

    Breit, Andreas; Wicht, Kristina; Boekhoff, Ingrid; Glas, Evi; Lauffer, Lisa; Mückter, Harald; Gudermann, Thomas

    2016-07-01

    Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction.

  10. Detection and evaluation of AmpC enzyme of multidrug-resistant acinetobacter baumanii%多药耐药鲍氏不动杆菌AmpC酶的检测及评价

    Institute of Scientific and Technical Information of China (English)

    赵水娣; 冯旰珠; 雷忠英

    2011-01-01

    目的 探讨临床分离的多药耐药鲍氏不动杆菌AmpC酶活性及基因型.方法 AmpC酶活性检测采用三维法、三联纸片法;基因检测采用PCR法.结果 20株多药耐药鲍氏不动杆菌ampC(染色体型)基因阳性14株,阳性率70.0%,ampC(质粒型)基因阳性1株,阳性率5.0%,总阳性率75.0%;三联纸片法阳性率80.0%,三维法阳性率65.0%.结论 三联纸片法、三维法与基因检测结果符合率接近,三联纸片法优于经典三维法,且较基因检测法更简捷方便.%OBJECTIVE To analyze AmpC enzyme activity and its gene type of multidrug-resistant Acinetobacter baumanii isolated from the clinics.METHODS Three-dimensional test and triple combination paper disk method were used for detection of AmpC enzyme activity, and polymerase chain reaction (PCR) was used for detection of the gene type.RESULTS Among 20 multi-drug resis tance A.baumanii strains, AmpC gene positive strains of chromosomal pattern were 14 stains with positive rate 70.0%, and positive strain of plasmid pattern was 1 strain with positive rate 5.0%.The total positive rate was 75.0%.The positive rate was 80.0% through triple combination paper disk method and it was 65.0% through three-dimensional test.CONCLUSION The detection results of triple combination paper disk method and three-dimensional test have almost the same coincidence rate compared with gene detection method.And triple combination paper disk method is better than traditional threedimensional test and is more simple and convenient compared with gene detection.

  11. AmpC酶弗劳地枸橼酸杆菌的耐药基因研究%Study on resistance gene of AmpC β-lactamase in Citrobacter freundii

    Institute of Scientific and Technical Information of China (English)

    曾红莲; 赵东明; 徐韫健; 张晓坤

    2011-01-01

    Aim To investigate the drug resistance gene of AmpC β-Lactamase from ten Citrobacter freundii.Methods -lactamases of ten strains was extracted by ultrasonic crushing,and AmpC -lactamases was tested with Cefoxitin in three dimensional test,the genotype of AmpC were amplified by multiple PCR and PCR products were sequenced subsequently. Drug sensitivity of ten strains were determined. Results There were 4 strains positive in three-dimensional test.The genotype of β-lactamases were not detected in CMY-G1 (1/10),CMY-G2 (2/10),FOX (1/10),CIT(2/10),ACT,DHA and MOX genes in ten strains. The 1 146bp DNA fragment of CMY-type AmpC β-Lactamase gene sequence shared 99 % amino acid identity with blaCMY that already appeared in Guangzhou. The resistant strains overexpressing AmpC β-lactamases were multi-drug resistant. Conclusion The surveillance of overexpressing AmpC β-lactamases in Citrobacter freundii should be emphasized so as to choose effective antibiotic for dealing with AmpC β-lactamases producing strains.%目的 对广州医学院第一附属医院临床分离的10株弗劳地枸橼酸杆菌进行AmpC酶的耐药基因研究.方法 用超声破碎法提取10株菌的β-内酰胺酶粗提物,进行三维试验;提取10株细菌的总DNA,PCR扩增CMY-G1、CMY-G2、FOX、ACT、DHA、MOX、CIT耐药基因并进行测序;对菌株进行MIC药物敏感试验.结果 三维试验阳性的有4株,β-内酰胺酶基因的检出率:CMY-G1(1/10)、CMY-G2(2/10)、FOX(1/10)、CIT(2/10),ACT、DHA和MOX未检出;CMY基因经全序列测定得到1 146bp的基因片段,与广州地区报道的CMY基因有99%同源;AmpC酶阳性的菌株茵呈多重耐药现象.结论 加强菌弗劳地枸橼酸杆菌产AmpC酶的监测,以选择合适的抗生素应对产酶株.

  12. Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A.

    Science.gov (United States)

    Faucheux, N; Nagel, M D

    2002-06-01

    Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling. PMID:12013176

  13. Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses

    NARCIS (Netherlands)

    Pacholewicz, Ewa; Liakopoulos, Apostolos; Swart, Arno; Gortemaker, Betty; Dierikx, Cindy; Havelaar, Arie; Schmitt, Heike

    2015-01-01

    Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the conce

  14. Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses

    NARCIS (Netherlands)

    Pacholewicz, Ewa; Liakopoulos, Apostolos; Swart, Arno; Gortemaker, Betty; Dierikx, Cindy; Havelaar, Arie; Schmitt, Heike

    2015-01-01

    Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the concentr

  15. Variation in genes coding for AMP-activated protein kinase (AMPK) and breast cancer risk in the European Prospective Investigation on Cancer (EPIC)

    NARCIS (Netherlands)

    Campa, Daniele; Claus, Rainer; Dostal, Lucie; Stein, Angelika; Chang-Claude, Jenny; Meidtner, Karina; Boeing, Heiner; Olsen, Anja; Tjonneland, Anne; Overvad, Kim; Rodriguez, Laudina; Bonet, Catalina; Sanchez, Maria-Jose; Amiano, Pilar; Huerta, Jose Maria; Barricarte, Aurelio; Khaw, Kay-Tee; Wareham, Nicholas; Travis, Ruth C.; Allen, Naomi E.; Trichopoulou, Antonia; Bamia, Christina; Benetou, Vassiliki; Palli, Domenico; Agnoli, Claudia; Panico, Salvatore; Tumino, Rosario; Sacerdote, Carlotta; van Kranen, Henk; Bueno-de-Mesquita, H. Bas; Peeters, Petra H. M.; van Gils, Carla H.; Lenner, Per; Sund, Malin; Lund, Eiliv; Gram, Inger Torhild; Rinaldi, Sabina; Chajes, Veronique; Romieu, Isabelle; Engel, Pierre; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Francoise; Siddiq, Afshan; Riboli, Elio; Canzian, Federico; Kaaks, Rudolf

    2011-01-01

    AMP-activated protein kinase (AMPK) is an energy sensing/signalling intracellular protein which is activated by an increase in the cellular AMP:ATP ratio after ATP depletion. Once activated, AMPK inhibits fatty acid synthesis and the Akt-mTOR pathway, and activates the p53-p21 axis. All these molecu

  16. Occurrence and characteristics of extended-spectrum-ß-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses

    NARCIS (Netherlands)

    Dierikx, C.M.; Duijkeren, van E.; Schoormans, A.; Essen-Zandbergen, van A.; Veldman, K.T.; Kant, A.; Huijsdens, X.; Zwaluw, van der K.; Wagenaar, J.A.; Mevius, D.J.

    2012-01-01

    To investigate the occurrence and characteristics of extended-spectrum -lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. Between October 2007 and August

  17. Constitutive high expression of chromosomal beta-lactamase in Pseudomonas aeruginosa caused by a new insertion sequence (IS1669) located in ampD

    DEFF Research Database (Denmark)

    Bagge, N.; Ciofu, O.; Hentzer, Morten;

    2002-01-01

    The expression of chromosomal AmpC beta-lactamase in Pseudomonas aeruginosa is negatively regulated by the activity of an amidase, AmpD. In the present study we examined resistant clinical P. aeruginosa strains and several resistant variants isolated from in vivo and in vitro biofilms for mutatio...

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

    Science.gov (United States)

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

    2014-10-01

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

  19. Molecular characterization of a defense-related AMP-binding protein gene, OsBIABP1, from rice

    Institute of Scientific and Technical Information of China (English)

    Xin-chun ZHANG; Xin YU; Hui-juan ZHANG; Feng-ming SONG

    2009-01-01

    We cloned and characterized a rice gene OsBIABPI encoding an AMP-binding protein. The full-length cDNA of OsBIABP1 is 1912-bp long and is predicted to encode a 558-aa protein. OsBIABP1 contains a typical AMP-binding signature motif and shows high similarity to members of AMP-binding protein family. OsBIABP1 is expressed in stems, leaves and flowers of rice plants, but is not expressed, or expressed at a very low level, in rice roots. The expression of OsBIABP1 was induced by some defense-related signal molecules, e.g., salicylic acid (SA), benzothiadiazole, jasmonic acid (JA), and 1-amino cyclopropane-1-carboxylic acid, which mediate SA-and JA/ethylene (ET)-dependent defense signaling pathways, respectively. Furthermore, the expression of OsBIABP1 is activated by the infection of Magnaporthe oryzae, and the induced expression is quicker and stronger during early stages of pathogenesis in incompatible interaction than that in compatible interaction between rice and M. oryzae. Our results suggest that OsBIABP1 may be a defense-related AMP-binding protein that is involved in the regulation of defense re-sponse through SA and/or JA/ET signaling pathways.

  20. Virodhamine and CP55,940 modulate cAMP production and IL-8 release in human bronchial epithelial cells

    NARCIS (Netherlands)

    Gkoumassi, E.; Dekkers, B. G. J.; Droege, M. J.; Elzinga, C. R. S.; Schmidt, M.; Meurs, H.; Zaagsma, J.; Nelemans, S. A.

    2007-01-01

    Background and purpose: We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells. Experimental approach: Human bronchial e

  1. The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx

    Directory of Open Access Journals (Sweden)

    Schlatterer Christina

    2006-06-01

    Full Text Available Abstract Background cAMP-induced Ca2+-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca2+-stores: acidic stores and the endoplasmic reticulum (ER. The acidic stores may comprise the contractile vacuole network (CV, the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca2+-store was investigated. Results Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release Ca2+. Release of Ca2+ was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca2+-transport and Ca2+-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca2+-compartment we wanted to know whether or not it takes part in cAMP-induced Ca2+-influx. We made use of the LvsA--mutant expected to display reduced Ca2+-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca2+-influx into whole cells. Conclusion The contractile vacuoles in Dictyostelium represent a highly efficient acidic Ca2+-store that is required for cAMP-induced Ca2+-influx.

  2. Oral glucose ingestion attenuates exercise-induced activation of 5'-AMP-activated protein kinase in human skeletal muscle

    DEFF Research Database (Denmark)

    Åkerström, Thorbjörn; Birk, Jesper Bratz; Klein, Ditte Kjærsgaard;

    2006-01-01

    5'-AMP-activated protein kinase (AMPK) has been suggested to be a 'metabolic master switch' regulating various aspects of muscle glucose and fat metabolism. In isolated rat skeletal muscle, glucose suppresses the activity of AMPK and in human muscle glycogen loading decreases exercise-induced AMPK...

  3. CRP-cyclic AMP dependent inhibition of the xylene-responsive σ(54-promoter Pu in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Yuan-Tao Zhang

    Full Text Available The expression of σ(54-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP, a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1 of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed.

  4. Characterization of cAMP accumulation mediated by three α1—adrenoceptor subtypes in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    SONGYao; HUANGYan; DongEr-Dan; HANQi-De; ZHANGYou-Yi

    2003-01-01

    AIM:To investigate the characterization of cAMP response mediated by α1-adrenoceptor (α1-AR) subtypes in HEK293 cells. METHODS:(1) Full-length cDNA encoding three α1-AR subtypes were transfected into HEK293 cells by the calcium phosphate precipitation method, respectively. (2) The densities of α1-AR subtypes expressed in HEK293 cells were measured by radioligand binding assay. (3)cAMP accumulation was measured by [3H] adenine prelabeling method. RESULTS: (1)Activation of each of three subtypes resulted in an increase of cAMP accumulation in HEK293 cells in a dose-dependent manner, which was inhibited by selective α1-AR antagonist prazosin. (2) Comparing the pharmacological property, the maximal responses of α1A-AR to agonists were the most potent, while the sensitivity of α1-AR subtypes to norepinephrine(NE) was the highest. CONCLUSION: Each of three α1-AR subtypes can mediate cAMP accumulation in HEK293 cell line, and there are differences in pharmacological property.

  5. Mass Spectrometry Imaging Reveals Elevated Glomerular ATP/AMP in Diabetes/obesity and Identifies Sphingomyelin as a Possible Mediator

    Directory of Open Access Journals (Sweden)

    Satoshi Miyamoto

    2016-05-01

    Full Text Available AMP-activated protein kinase (AMPK is suppressed in diabetes and may be due to a high ATP/AMP ratio, however the quantitation of nucleotides in vivo has been extremely difficult. Via matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI to localize renal nucleotides we found that the diabetic kidney had a significant increase in glomerular ATP/AMP ratio. Untargeted MALDI-MSI analysis revealed that a specific sphingomyelin species (SM(d18:1/16:0 accumulated in the glomeruli of diabetic and high-fat diet-fed mice compared with wild-type controls. In vitro studies in mesangial cells revealed that exogenous addition of SM(d18:1/16:0 significantly elevated ATP via increased glucose consumption and lactate production with a consequent reduction of AMPK and PGC1α. Furthermore, inhibition of sphingomyelin synthases reversed these effects. Our findings suggest that AMPK is reduced in the diabetic kidney due to an increase in the ATP/AMP ratio and that SM(d18:1/16:0 could be responsible for the enhanced ATP production via activation of the glycolytic pathway.

  6. Slow mitochondrial repair of 5'-AMP renders mtDNA susceptible to damage in APTX deficient cells

    DEFF Research Database (Denmark)

    Akbari, Mansour; Sykora, Peter; Bohr, Vilhelm A

    2015-01-01

    Aborted DNA ligation events in eukaryotic cells can generate 5'-adenylated (5'-AMP) DNA termini that can be removed from DNA by aprataxin (APTX). Mutations in APTX cause an inherited human disease syndrome characterized by early-onset progressive ataxia with ocular motor apraxia (AOA1). APTX...

  7. Bronchial responsiveness to adenosine 5 '-monophosphate (AMP) and methacholine differ in their relationship with airway allergy and baseline FEV

    NARCIS (Netherlands)

    De Meer, G; Heederik, D; Postma, DS

    2002-01-01

    Bronchial hyperresponsiveness (BHR) and inflammation are central hallmarks of asthma. Studies in patients with asthma suggest that BHR to adenosine 5'-monophosphate (AMP) is a better marker of bronchial inflammation than BHR to methacholine. The association between markers of airway inflammation and

  8. Glutamate Acts as a Key Signal Linking Glucose Metabolism to Incretin/cAMP Action to Amplify Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Ghupurjan Gheni

    2014-10-01

    Full Text Available Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

  9. AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Wojtaszewski, Jørgen; Richter, Erik

    2009-01-01

    In skeletal muscle, the contraction-activated heterotrimeric 5'-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved...

  10. Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells in Dictyostelium discoideum

    Indian Academy of Sciences (India)

    Ira N Feit; Erika J Medynski; Michael J Rothrock

    2001-06-01

    A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH3) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.

  11. Imaging cAMP-specific phosphodiesterase-4 in human brain with R-[11C]rolipram and positron emission tomography

    International Nuclear Information System (INIS)

    Evidence of disruptions in cAMP-mediated signaling in several neuropsychiatric disorders has led to the development of R-[11C]rolipram for imaging phosphodiesterase-4 (PDE4) enzymes with positron emission tomography (PET). The high-affinity PDE4 inhibitor rolipram was previously reported to have an antidepressant effect in humans. PDE4 is abundant in the brain, and it hydrolyzes cAMP produced following stimulation of various neurotransmitter systems. PDE4 is regulated by intracellular cAMP levels. This paper presents the first PET study of R-[11C]rolipram in living human brain. Consistent with the wide distribution of PDE4, high radioactivity retention was observed in all regions representing the gray matter. Rapid metabolism was observed, and kinetic analysis demonstrated that the data fit in a two-tissue compartment model. R-[11C]Rolipram is thus suitable for imaging PDE4 and possibly cAMP signal transduction in the living human brain with PET. (orig.)

  12. Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells

    NARCIS (Netherlands)

    Xie, Yufen; Awonuga, Awoniyi; Liu, Jian; Rings, Edmond; Puscheck, Elizabeth Ella; Rappolee, Daniel A.

    2013-01-01

    The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id) 2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the

  13. COUPLING OF THE ANTIVIRAL DRUG ARA-AMP TO LACTOSAMINATED ALBUMIN LEADS TO SPECIFIC UPTAKE IN RAT AND HUMAN HEPATOCYTES

    NARCIS (Netherlands)

    JANSEN, RW; KRUIJT, JK; VANBERKEL, TJC; MEIJER, DKF

    1993-01-01

    We covalently coupled 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) to the carrier molecule lactosaminated human serum albumin using a water-soluble carbodiimide with a two-step conjugation method (pH 4.5 and pH 7.5) instead of the commonly used single-step conjugation at pH 7.5. This

  14. Coexistence of plasmid-mediated quinolone resistance determinants and AmpC-Beta-Lactamases in Escherichia coli strains in Egypt.

    Science.gov (United States)

    Abd El-Aziz, N K; Gharib, A A

    2015-01-01

    Three kinds of plasmid—mediated quinolone resistance (PMQR) determinants (qnr genes, qepA and aac(6')—Ib—cr) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants among AmpC—producing E. coli strains in food—producing animals and animal by—products in Egypt, twenty—nine E. coli strains were tested for their susceptibilities to antimicrobials and screened for PMQR determinants and AmpC Beta lactamases using PCR and plasmid profiling. It was found that qnr genes being detected alone or in combination with qepA or aac(6')—Ib—cr genes in 11 (37.9%) strains comprising 9 for qnrA and only one for both qnrB and qnrS. Moreover, qepA and aac(6')—Ib—cr were detected in 41.38% and 3.45% of E. coli strains, respectively. The ampC β—lactamase genes were detected in 75.86 % of all strains and in 100% and 53.3% of the PMQR determinant—positive and negative strains, respectively. In several cases, plasmid profiling of E. coli strains exhibiting the coexistence of both PMQR determinants and ampC genes on a single plasmid as a first report in Egypt that may contribute to rapid spread and increase in bacterial resistance, which is important to public health concern. PMID:26475385

  15. Cytoplasmic and membranous CD24 marker expression has indirect correlation with cAMP/cGMP ratio

    Directory of Open Access Journals (Sweden)

    Hossein Goudarzi

    2014-02-01

    Full Text Available Background- CD24 is a cell adhesion molecule that has been implicated in metastatic tumor progression cells. Our aim was clarify correlation between CD24 expression and cAMP:cGMP ratio in murine colorectal cancer cell line (CT26 after using cholera toxin.Materials and Methods- The CT26 cells were cultured in microtubes for assaying cAMP and cGMP; also the cells were cultured in flasks for assaying cytoplasmic and membranous CD24 expression. The Real-Time PCR was done for cDNA that was synthesized from CT26 cells’ mRNA. Also, expression CD24 marker of cells was determined by Anti-CD24 antibody and Goat Anti-Rabbit IgG-FITC (flow cytometry.Results- The cholera toxin grew cAMP:cGMP ratio and it influenced cytoplasmic and membrane CD24 expression.Conclusion- There are indirect correlation between cAMP:cGMP ratio and CD24 expression.

  16. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    International Nuclear Information System (INIS)

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  17. IP{sub 3}-dependent intracellular Ca{sup 2+} release is required for cAMP-induced c-fos expression in hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenting; Tingare, Asmita; Ng, David Chi-Heng [Department of Pharmacology, University of Cambridge (United Kingdom); Johnson, Hong W.; Schell, Michael J. [Department of Pharmacology, Uniformed Services University, Bethesda (United States); Lord, Rebecca L. [Department of Biology, University of York (United Kingdom); Chawla, Sangeeta, E-mail: sangeeta.chawla@york.ac.uk [Department of Pharmacology, University of Cambridge (United Kingdom); Department of Biology, University of York (United Kingdom)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer cAMP-induced c-fos expression in hippocampal neurons requires a submembraneous Ca{sup 2+} pool. Black-Right-Pointing-Pointer The submembraneous Ca{sup 2+} pool derives from intracellular ER stores. Black-Right-Pointing-Pointer Expression of IP{sub 3}-metabolizing enzymes inhibits cAMP-induced c-fos expression. Black-Right-Pointing-Pointer SRE-mediated and CRE-mediated gene expression is sensitive to IP{sub 3}-metabolizing enzymes. Black-Right-Pointing-Pointer Intracellular Ca{sup 2+} release is required for cAMP-induced nuclear translocation of TORC1. -- Abstract: Ca{sup 2+} and cAMP are widely used in concert by neurons to relay signals from the synapse to the nucleus, where synaptic activity modulates gene expression required for synaptic plasticity. Neurons utilize different transcriptional regulators to integrate information encoded in the spatiotemporal dynamics and magnitude of Ca{sup 2+} and cAMP signals, including some that are Ca{sup 2+}-responsive, some that are cAMP-responsive and some that detect coincident Ca{sup 2+} and cAMP signals. Because Ca{sup 2+} and cAMP can influence each other's amplitude and spatiotemporal characteristics, we investigated how cAMP acts to regulate gene expression when increases in intracellular Ca{sup 2+} are buffered. We show here that cAMP-mobilizing stimuli are unable to induce expression of the immediate early gene c-fos in hippocampal neurons in the presence of the intracellular Ca{sup 2+} buffer BAPTA-AM. Expression of enzymes that attenuate intracellular IP{sub 3} levels also inhibited cAMP-dependent c-fos induction. Synaptic activity induces c-fos transcription through two cis regulatory DNA elements - the CRE and the SRE. We show here that in response to cAMP both CRE-mediated and SRE-mediated induction of a luciferase reporter gene is attenuated by IP{sub 3} metabolizing enzymes. Furthermore, cAMP-induced nuclear translocation of the CREB coactivator TORC1 was inhibited

  18. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  19. Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP signaling to inhibit steroidogenesis in human granulosa cells

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    HoYuen Basil

    2009-10-01

    Full Text Available Abstract Background Obesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells. Methods The effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG cells was examined by Enzyme linked immunosorbent assay (ELISA. The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner. Results and Conclusion In the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059 and p38 (SB203580 inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone

  20. Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure

    DEFF Research Database (Denmark)

    Torp, M; Brønd, L; Hadrup, N;

    2007-01-01

    receptor type-1 (AT(1)) blockade with losartan. AIM: In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT(1) receptor blockade on this system. METHOD: CHF was induced by ligation of the left anterior descending coronary...... artery (LAD). SHAM-operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day(-1) i.p.). RESULTS: CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10......(-6) m) stimulated cAMP levels were significantly increased in CHF rats (25.52 +/- 4.49 pmol cAMP microg(-1) protein, P Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 +/- 1.93 fmol...

  1. Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli

    International Nuclear Information System (INIS)

    The cDNA for the bovine type I regulatory subunit of cAMP-dependent protein kinase has been inserted into the expression vector pUC7. When E. coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 2-4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-N3-[32P]-cAMP following transfer from SDS polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of IPTG. R-subunit accumulated in large amounts only in the stationary phase of growth. The addition of IPTG during the log phase of growth actually blocked the accumulation of R-subunit. The soluble, dimeric R-subunit was purifided to homogeneity by affinity chromatography. This R-subunit bound 2 mol cAMP/mol R monomer, reassociated with C-subunit to form cAMP-dependent holoenzyme, and migrated as a dimer on SDS polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties the expressed protein was indistinguishable from R/sup I/ purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z gene of the vector. The NH2-terminal sequence was confirmed by amino acid sequencing

  2. Changes in ganglioside contents, plasma sialic acid and cAMP levels in experimental hepatoma in mice

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Abstract The present sutdy was designed to assess whether changes in glycolipids and cyclic AMP contents might serve as markers for the diagnosis of malignancy in the liver. The experimental model was a transplantable murine hepatoma. Experimental mice were divided into three groups:(1) a therapeutic group. which had been transplanted with hepatoma and treated with the antimetabolism drug 5-flurouracil(0.2mg/day i.p).(2) a control group, which had been transplanted with hepatoma and treated with 0.2ml 0.9% NaCl day and (3) a normal group of mice . The ganglioside and cAMP contents in the hepatoma tissue, plasma cAMP, total and lipid-bound sialic acid levels and red blood cell memebrane sialic acid levels were determined. Results showed that the ganglioside content, total and lipid-bound sialic acid levels in the control group were significantly higher than those in the livers of normal mice(P<0.01) while these respective values in the therapeutic group were significantly lower than those in the control group(P<0.01).The cAMP levels of tumor tissues and plasma in the control group were lower than those in normal mice. No significant difference in red blood cell membrane sialic acid content was observed between the therapeutic and control groups though levels for both were higher than those in normal mice. These results indicate that ganglioside ocntent and sialic acid levels in hepatoma tissues were significantly elevated, and cAMP levels in hepatoma tissues were significantly decreased during proliferation and abnormal differentiation. Key words transplantable hepatoma, gangliosides , sialic acid, cyclic adenosine mono-phosphate, 5-flurouracil 摘自Molecular and cellular biochemistry, 2000; Vol 207,29~33 该杂志摘要被美国科学引证索引(SCI)收录

  3. Microbiological characterization of plasmid-mediated AmpC ß-lactamases and E. coli hyperproducers: how and why ?

    Directory of Open Access Journals (Sweden)

    Annibale Raglio

    2010-03-01

    Full Text Available The aim of this study is the evaluation of phenotypic method for the detection of plasmid-mediated AmpC producing Enterobacteriaceae by agar diffusion.We developed a phenotypic method with double disk test (CLSI and evaluation of synergism between Cloxacillin and/or Boronic Acid with cefotaxime and ceftazidime and cefepime with amoxicillin/clavulanic acid. As reference method for AmpC detection we used a multiplex PCR according to Perez-Perez. Among 7476 Enterobacteriaceae we detected 45 strains: 37 (82.2% plasmid-mediated AmpC producers, 6 (13.3% E. coli hyperproducers and 2 E. coli (4.5% positive for both.The AmpC phenotypic test was positive for all the isolates, showing a typical ghost zone between cloxacillin and cephalosporins or boronic acid and cephalosporins.The AmpC multiplex PCR confirmed that 28 P. mirabilis and 7 E. coli harboured a gene belonging to the bla-CMY-LAT family. Sequencing defined the presence of CMY-16 in all P. mirabilis, CMY-2 in E. coli, DHA-1 in 3 K. pneumoniae and FOX in 1 K. pneumoniae and allowed us to identify eight strains as E. coli hyperproducer: six E. coli yielded no amplicon and 2 were also producer of CMY-2. In this study the phenotypic method showed a sensitivity and a specificity of 100%.Waiting for the indication of international authorities, we think this phenotypic screening method could be useful in the routine of microbiological laboratories.

  4. Stress and glucocorticoids impair memory retrieval via β2-adrenergic, Gi/o-coupled suppression of cAMP signaling.

    Science.gov (United States)

    Schutsky, Keith; Ouyang, Ming; Castelino, Christina B; Zhang, Lei; Thomas, Steven A

    2011-10-01

    Acute stress impairs the retrieval of hippocampus-dependent memory, and this effect is mimicked by exogenous administration of stress-responsive glucocorticoid hormones. It has been proposed that glucocorticoids affect memory by promoting the release and/or blocking the reuptake of norepinephrine (NE), a stress-responsive neurotransmitter. It has also been proposed that this enhanced NE signaling impairs memory retrieval by stimulating β(1)-adrenergic receptors and elevating levels of cAMP. In contrast, other evidence indicates that NE, β(1), and cAMP signaling is transiently required for the retrieval of hippocampus-dependent memory. To resolve this discrepancy, wild-type rats and mice with and without gene-targeted mutations were stressed or treated with glucocorticoids and/or adrenergic receptor drugs before testing memory for inhibitory avoidance or fear conditioning. Here we report that glucocorticoids do not require NE to impair retrieval. However, stress- and glucocorticoid-induced impairments of retrieval depend on the activation of β(2) (but not β(1))-adrenergic receptors. Offering an explanation for the opposing functions of these two receptors, the impairing effects of stress, glucocorticoids and β(2) agonists on retrieval are blocked by pertussis toxin, which inactivates signaling by G(i/o)-coupled receptors. In hippocampal slices, β(2) signaling decreases cAMP levels and greatly reduces the increase in cAMP mediated by β(1) signaling. Finally, augmenting cAMP signaling in the hippocampus prevents the impairment of retrieval by systemic β(2) agonists or glucocorticoids. These results demonstrate that the β(2) receptor can be a critical effector of acute stress, and that β(1) and β(2) receptors can have quite distinct roles in CNS signaling and cognition.

  5. Cellular effects of beta-adrenergic and of cAMP stimulation on potassium transport in rat alveolar epithelium.

    Science.gov (United States)

    Saumon, G; Basset, G; Bouchonnet, F; Crone, C

    1989-07-01

    Alveolar fluid absorption is greatly enhanced by cAMP and by beta-adrenergic agonists via an increase in Na+ transport. Little is known about K+ homeostasis under these circumstances. We studied K+ transport across alveolar epithelium in isolated perfused rat lungs stimulated either by dibutyryl-cAMP or isoproterenol. K+ fluxes and the apparent permeability of 86Rb across the epithelium (alveoli to plasma) were interpreted according to a model involving two types of cells, B and L, distinguished by the location of Na+-K+-ATPases (basal and luminal). Water is considered to be absorbed by B cells in a solute-coupled process energized by a basolateral Na+-K+-ATPase that is stimulated by isoproterenol and cAMP. K+ transport out of the alveoli is due to the activity of a Na+-K+-ATPase located in the apical membrane of L cells. In the present study net transport rate of K+ was -0.5 +/- 0.15 nmol/s, n = 20 (out of alveoli) in control conditions. When the epithelium was stimulated by dibutyryl-cAMP (10(-4) mol/l) net absorption of K+ reversed to net 'secretion' into alveoli (3.2 +/- 0.31 nmol/s), fluid absorption was not stimulated. K+ 'secretion' was abolished by apical Ba2+, indicating it was due to opening of apical K+ channels. Basolateral ouabain reversed net K+ 'secretion' to net absorption indicating that K+ entry into alveoli was dependent on activity of B cell basolateral Na+-K+-ATPase (masking simultaneous K+ removal by apical L cell Na+-K+-pump). When larger concentrations of dibutyryl-cAMP (10(-3) mol/l) or when isoproterenol were used to stimulate the epithelium there was a tripling of fluid absorption.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2571117

  6. Emergence of Escherichia coli Producing Extended-Spectrum AmpC beta-lactamases (ESAC in animals

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    Marisa eHaenni

    2014-02-01

    Full Text Available In both humans and animals, the spread of Extended-Spectrum -Lactamases (ESBL/AmpC producers has become a major issue, particularly due to the plasmidic dissemination of most of these genes. Besides, over-expression of the chromosomal ampC gene was largely reported in human and animal Enterobacteriaceae and, more recently, modifications within the coding region of the ampC gene (encoding Extended-spectrum AmpC -lactamases (ESACs were shown to be responsible for an hydrolysis spectrum expanded to oxyiminocephalosporins in humans. In this study, among 6765 cattle E. coli isolates, 28 (0.37% isolates harboring a reduced susceptibility to cefepime (MICs ranging from 0.5 to 12 g/ml were investigated as presumptive ESACs producers. Highly conserved mutations in the promoter/attenuator region were identified at positions -88, -82, -42, -18, -1 and +58. Using sequencing and cloning experiments, amino acid substitutions of the AmpC beta-lactamase were characterized at positions 287 (mostly S287N, but also S287C, 292 (A292V and 296 (H296P, similarly to data reported in humans. Interestingly, those cattle ESAC-producing E. coli isolates predominantly belonged to the Clonal Complex (CC23, thus mirroring what has been described in humans. The driving forces for the selection of ESACs in animals are unknown, and their prevalence needs to be further investigated in the different animal sectors. Considering the over-representation of ESAC-producing E. coli belonging to CC23 in both humans and animals, exchanges of ESAC producers between the two populations may have occurred as well. To our best knowledge, this study is the first report of ESACs in animals worldwide, which should be considered an emerging mechanism contributing to the resistance to extended-spectrum cephalosporins in the animal population.

  7. β2-Agonist induced cAMP is decreased in asthmatic airway smooth muscle due to increased PDE4D.

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    Thomas Trian

    Full Text Available BACKGROUND AND OBJECTIVE: Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. OBJECTIVE: To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. METHODS: We examined β(2-adrenergic (β(2AR receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase stimulated cAMP production in asthmatic cultured ASM (n = 15 and non-asthmatic ASM (n = 22. Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. RESULTS: In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. CONCLUSION: Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be "hard-wired" into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.

  8. Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes.

    Science.gov (United States)

    Plée-Gautier, E; Grober, J; Duplus, E; Langin, D; Forest, C

    1996-09-15

    Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.

  9. cAMP blocks MAPK activation and sclerotial development via Rap-1 in a PKA-independent manner in Sclerotinia sclerotiorum.

    Science.gov (United States)

    Chen, Changbin; Dickman, Martin B

    2005-01-01

    Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen-activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)-independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap-1 or Ras is involved in cAMP action. The Rap-1 inhibitor, GGTI-298, restored MAPK activation in the presence of cAMP, further suggesting that Rap-1 is responsible for cAMP-dependent MAPK inhibition. Importantly, inhibition of Rap-1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA-independent cAMP signalling pathway. Cross-talk between these two pathways is mediated by Rap-1. PMID:15612936

  10. Phospholipase D1 mediates AMP-activated protein kinase signaling for glucose uptake.

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    Jong Hyun Kim

    Full Text Available BACKGROUND: Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here, we found that AMPK-induced phospholipase D1 (PLD1 activation is required for (14C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT stimulates PLD activity, while AMPK-dominant negative (DN inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in (14C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of (14C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA, which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate (14C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator regulate (14C-glucose uptake and cell surface glucose transport (GLUT 4 through ERK stimulation by AMPK-mediated PLD1 activation. CONCLUSIONS/SIGNIFICANCE: These results

  11. Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.

    Science.gov (United States)

    Worrell, Roger T; Best, Alison; Crawford, Oscar R; Xu, Jie; Soleimani, Manoocher; Matthews, Jeffrey B

    2005-10-01

    Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is

  12. Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

    Energy Technology Data Exchange (ETDEWEB)

    Trehan, I.; Beadle, B.M.; Shoichet, B.K. (NWU)

    2010-03-08

    {beta}-Lactamases hydrolyze {beta}-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. {beta}-Lactams that contain a bulky 6(7){alpha} substituent, such as imipenem and moxalactam, actually inhibit serine {beta}-lactamases and are widely used for this reason. Although mutant serine {beta}-lactamases have arisen that hydrolyze {beta}-lactamase resistant {beta}-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine {beta}-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7){alpha} substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C {beta}-lactamases (Asn132 in class A {beta}-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C {beta}-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 {yields} Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 {angstrom}. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces {beta}-lactams with 6(7){alpha

  13. Prevalence and characterization of ESBL- and AmpC-producing Enterobacteriaceae on retail vegetables.

    Science.gov (United States)

    van Hoek, Angela H A M; Veenman, Christiaan; van Overbeek, Wendy M; Lynch, Gretta; de Roda Husman, Ana Maria; Blaak, Hetty

    2015-07-01

    In total 1216 vegetables obtained from Dutch stores during 2012 and 2013 were analysed to determine the prevalence of 3rd-generation cephalosporin (3GC) resistant bacteria on soil-grown fresh produce possibly consumed raw. Vegetables grown conventionally and organically, from Dutch as well as foreign origin were compared. Included were the following vegetable types; blanched celery (n=192), bunched carrots (n=190), butterhead lettuce (n=137), chicory (n=96), endive (n=188), iceberg lettuce (n=193) and radish (n=120). Overall, 3GC-resistant Enterobacteriaceae were detected on 5.2% of vegetables. Based on primary habitat and mechanism of 3GC-resistance, these bacteria could be divided into four groups: ESBL-producing faecal species (Escherichia coli, Enterobacter spp.), AmpC-producing faecal species (Citrobacter freundii, Enterobacter spp.), ESBL-producing environmental species (Pantoea spp., Rahnella aquatilis, Serratia fonticola), and AmpC-producing environmental species (Cedecca spp., Hafnia alvei, Pantoea spp., Serratia plymuthica), which were detected on 0.8%, 1.2%, 2.6% and 0.4% of the vegetables analysed, respectively. Contamination with faecal 3GC-resistant bacteria was most frequently observed in root and bulb vegetables (average prevalence 4.4%), and less frequently in stem vegetables (prevalence 1.6%) and leafy greens (average prevalence 0.6%). In Dutch stores, only four of the included vegetable types (blanched celery, bunched carrots, endive, iceberg lettuce) were available in all four possible variants: Dutch/conventional, Dutch/organic, foreign/conventional, foreign/organic. With respect to these vegetable types, no statistically significant difference was observed in prevalence of 3GC-resistant Enterobacteriaceae between country of origin or cultivation type (5.2%, 5.7%, 5.7% and 3.3%, respectively). Vegetables consumed raw may be a source of dissemination of 3GC-resistant Enterobacteriaceae and their resistance genes to humans. The magnitude of the

  14. Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

    Science.gov (United States)

    Walther-Rasmussen, J; Johnsen, A H; Høiby, N

    1999-07-01

    AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms. PMID:10406957

  15. Structure-based Design and In-Parallel Synthesis of Inhibitors of AmpC b-lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Tondi, D.; Powers, R.A.; Negri, M.C.; Caselli, M.C.; Blazquez, J.; Costi, M.P.; Shoichet, B.K. (NWU)

    2010-03-08

    Group I {beta}-lactamases are a major cause of antibiotic resistance to {beta}-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic {beta}-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I {beta}-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of {beta}-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K{sub i} 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K{sub i} values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 {angstrom} resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from {beta}-lactams but

  16. Dual effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109

    Institute of Scientific and Technical Information of China (English)

    Hong-Mei Wang; Nai-Gang Zheng; Jing-Lan Wu; Cui-Cui Gong; Yi-Ling Wang

    2005-01-01

    AIM: To investigate the effects of 8-Br-cAMP on differentiation and apoptosis of human esophageal cancer cell line Eca-109, and the related gene expression.METHODS: The cultured Eca-109 cells were divided into four groups: E1 group (co-cultured with 8-Br-cAMP for 24 h); E2 group (co-cultured with 8-Br-cAMP for 48 h); C1 group (treated without 8-Br-cAMP for 24 h); and C2 group (treated without 8-Br-cAMP for 48 h). The same concentration of cell suspension of each group was dropped separately onto the slides and nitrocellulose membranes (NCM). The biotin-labeled cDNA probes for c-myc, wild-type (wt) p53, bcl-2 and iNOS were prepared for in situ hybridization. The expressions of epidermal growth factor receptor (EGFR), p38 kinase, FAS, FasL and caspase-3 were detected using immunocytochemistry, and the NOS activity and the ratio of differentiated cells/proliferating cells were examined by cytochemistry. Immunocytochemistry, cytochemistry,and in situ hybridization were separately carried out on both slides and NCM specimens for each group. In addition, TUNEL was used to detect the cell apoptosis rate in each group.RESULTS: The apoptotic rate of E2 group was significantly higher compared to E1 group, while there was no difference in the ratio of differentiated cells/ proliferating cells between E1 and E2 groups. The signals of wt p53 and iNOS were markedly stronger, while the signals of c-myc and EGFR were obviously weaker in E1 group than those in C1 group (P<0.05). Moreover, the signals of wt p53, iNOS, p38 kinase, caspase-3 and NOS activity were significantly stronger, whereas, the signals of bcl-2, c-myc and Fas/FasL were markedly weaker in E2 group than those in C2 group (P<0.05). CONCLUSION: The differentiation and apoptosis of human esophageal cancer cell Eca-109 can be induced after 24- and 48-h treatment with 8-BrcAMP, respectively. Upregulation of wt p53, iNOS and downregulation of c-myc may be associated with differentiation and apoptosis of Eca-109 cells

  17. Genotypic and Phenotypic Detection of AmpC β-lactamases in Enterobacter spp. Isolated from a Teaching Hospital in Malaysia.

    Directory of Open Access Journals (Sweden)

    Fatin Izzati Mohd Khari

    Full Text Available The objective of this study was to determine the occurrence of chromosomal and plasmid-mediated β-lactamases (AmpC genes in a collection of Malaysian isolates of Enterobacter species. Several phenotypic tests for detection of AmpC production of Enterobacter spp. were evaluated and the agreements between tests were determined.Antimicrobial susceptibility profiles for 117 Enterobacter clinical isolates obtained from the Medical Microbiology Diagnostic Laboratory, University Malaya Medical Centre, Malaysia, from November 2012-February 2014 were determined in accordance to CLSI guidelines. AmpC genes were detected using a multiplex PCR assay targeting the MIR/ACT gene (closely related to chromosomal EBC family gene and other plasmid-mediated genes, including DHA, MOX, CMY, ACC, and FOX. The AmpC β-lactamase production of the isolates was assessed using cefoxitin disk screening test, D69C AmpC detection set, cefoxitin-cloxacillin double disk synergy test (CC-DDS and AmpC induction test.Among the Enterobacter isolates in this study, 39.3% were resistant to cefotaxime and ceftriaxone and 23.9% were resistant to ceftazidime. Ten (8.5% of the isolates were resistant to cefepime, and one isolate was resistant to meropenem. Chromosomal EBC family gene was amplified from 36 (47.4% E. cloacae and three (25% E. asburiae. A novel blaDHA type plasmid-mediated AmpC gene was identified for the first time from an E. cloacae isolate. AmpC β-lactamase production was detected in 99 (89.2% of 111 potential AmpC β-lactamase producers (positive in cefoxitin disk screening using D69C AmpC detection set. The detection rates were lower with CC-DDS (80.2% and AmpC induction tests (50.5%. There was low agreement between the D69C AmpC detection set and the other two phenotypic tests. Of the 40 isolates with AmpC genes detected in this study, 87.5%, 77.5% and 50.0% of these isolates were positive by the D69C AmpC detection set, CC-DDS and AmpC induction tests, respectively

  18. cAMP elevators inhibit LPS-induced IL-12 p40 expression by interfering with phosphorylation of p38 MAPK in Murine Peritoneal Macrophages

    Institute of Scientific and Technical Information of China (English)

    WEI; GUO; FENG; YI; BING; WANG; JIN; SONG; ZHANG; XING; YU; WANG; CHANG; LIN; LI; ZONG; LIANG; CHANG

    2002-01-01

    cAMP mediated signaling may play a suppressive role in immune response. We previously found thatthe cAMP-elevators (CTx and 8-Br-cAMP) inhibited IL-12, IL-la, IL-6 gene expression, but increasedthe transcriptional levels of IL-10 and IL-1Ra in LPS-treated murine peritoneal macrophages. The presentstudy examined a possible molecular mechanism involved in cAMP elevators-induced inhibition of IL-12 p40expression in response to LPS. Our data demonstrated that cAMP elevators downregulated IL-12 p40 mRNAexpression and IL-12 p70 production in murine peritoneal macrophages. Subsequent studies revealed thatcAMP-elevators blocked phosphorylation of p38 MAPK, but did not affect the activity of NF-κB bindingto IL-12 promoter (-136/-112). This is the first report that cAMP elevators inhibit LPS-induced IL-12production by a mechanism that is associated, at least in part, with p38-dependent inhibition by cAMPsignaling pathways.

  19. Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity

    Science.gov (United States)

    Ding, Lei; Zhang, Feng; Zhao, Ming-Xia; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-01-01

    Chemical stimulation of white adipose tissue (WAT) causes adipose afferent reflex (AAR) and sympathetic activation. This study is to investigate the effects of AAR on lipolysis and the mechanisms of attenuated lipolysis response to enhanced AAR in obesity. Obesity was caused by high-fat diet for 12 weeks in rats. AAR was induced by injection of capsaicin into inguinal WAT or electrical stimulation of epididymal WAT afferent nerve. AAR caused sympathetic activation, which was enhanced in obesity rats. AAR increased cAMP levels and PKA activity, promoted hormone sensitive lipase (HSL) and perilipin phosphorylation, and increased lipolysis in WAT, which were attenuated in obesity rats. PKA activity, cAMP, perilipin and β-adrenoceptor levels were reduced, while HSL was upregulated in adipocytes from obesity rats. In primary adipocytes, isoproterenol increased cAMP levels and PKA activity, promoted HSL and perilipin phosphorylation, and increased lipolysis, which were attenuated in obesity rats. The attenuated effects of isoproterenol in adipocytes from obesity rats were prevented by a cAMP analogue dbcAMP. The results indicate that reduced lipolysis response to enhanced AAR in obesity is attributed to the impaired activation of β-adrenoceptor-cAMP-PKA-HSL pathway. Increased cAMP level in adipocytes rectifies the attenuated lipolysis in obesity. PMID:27694818

  20. Effect of Increased Cyclic AMP Concentration on Muscle Protein Synthesis and Beta-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture

    Science.gov (United States)

    Young, R. B.; Vaughn, J. R.; Bridge, K. Y.; Smith, C. K.

    1998-01-01

    Analogies of epinephrine are known to cause hypertrophy of skeletal muscle when fed to animals. These compounds presumably exert their physiological action through interaction with the P-adrenergic receptor. Since the intracellular signal generated by the Beta-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation of cAMP by treatment with forskolin would alter muscle protein metabolism and P-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 micrometers forskolin for a total of three days. At the end of the treatment period, both the concentration of cAMP and the quantity of myosin heavy chain (MHC) were measured. Concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. In contrast, the quantity of MHC was increased approximately 50% above control cells at 0.2 micrometers forskolin, but exhibited a gradual decline at higher levels of forskolin so that the quantity of MHC in cells treated with 30 micrometers forskolin was not significantly different from controls. Curiously, the intracellular concentration of cAMP which elicited the maximum increase in the quantity of MHC was only 40% higher than cAMP concentration in control cells.

  1. Antifungal effect of antimicrobial peptides (AMPs LR14) derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage.

    Science.gov (United States)

    Gupta, Ruchi; Srivastava, Sheela

    2014-09-01

    The concern for food safety has led to an increased interest in the development of novel antimicrobials. Keeping this aim in mind, we have investigated the antifungal effect of antimicrobial peptides (AMPs LR14) produced by Lactobacillus plantarum strain LR/14 against four spoilage fungi, namely, Aspergillus niger, Rhizopus stolonifer, Mucor racemosus and Penicillium chrysogenum. Interestingly, all the four fungi were inhibited, suggesting that AMPs LR14 exhibited anti-fungal property. The peptides inhibited both, the spore germination and hyphal growth, however, the former stage was found to be more susceptible. The hyphal extensions were also inhibited in a dose-dependent manner. Viability test of treated spores confirmed the fungicidal activity of AMPs LR14. AMPs LR14 were also studied for the prevention of wheat grain spoilage under storage. Unhygienic conditions in damp godowns and store-houses, lead to loss of food grains and make them unfit for human consumption due to microbial deterioration. The treatment of wheat seeds with AMPs LR14 prevented fungal growth even after a prolonged storage under laboratory conditions for ∼2.5 years. The carbohydrate and protein content of the AMPs LR14-treated seeds denoted no significant loss, but the seed viability was affected as germination was retarded. Such studies have not been reported for any bacteriocin/AMP to the best of our knowledge.

  2. Determination of Residual Monomers in AM/AMPS Copolymer by High Performance Liquid Chromatography%高效液相色谱法测定AMPS/AM二元共聚物中残余微量单体含量

    Institute of Scientific and Technical Information of China (English)

    贾春革; 穆晓蕾; 刘希; 赵方园

    2013-01-01

    A high performance liquid chromatography for the analysis of residual monomers,2-acrylamido-2-methyl propane sulfonic acid(AMPS) and acrylamide(AM),in AM/AMPS copolymer was established.In order to reduce the viscosity of samples,the mixture of isopropanol and ethanol was used as the extractant to extract AMPS and AM from the aqueous solution of AM/AMPS copolymer.The effects of the wavelength and proportion of mobile phases on the separation were investigated to choose the optimal analysis conditions.The results show that the optimal conditions are as follow:chromatographic column ZORBAX SB-C18(4.0 mm×150 mm),volume ratio of methanol to water 85∶15 as the mobile phase,flow rate of the mobile phase 1.0 mL/min,UV absorption wavelength 220 nm,column temperature 27℃ and sample dosage 6 μL.The calibration curves of AMPS and AM were established.Their correlation coefficients were above 0.9995.The recoveries of the two components were in the range of 97.50%-100.62% and the relative standard deviations were in the range of 0.435 7%-1.2981%.The method is accurate,rapid and sensitive.%建立了一种同时测定2-丙烯酰胺基-2-甲基丙磺酸(AMPS)/丙烯酰胺(AM)二元共聚物中残余单体AMPS和AM含量的HPLC法;为降低试样的黏性,以异丙醇-乙醇的混合液为提取剂,将AMPS和AM从AMPS/AM二元共聚物的水溶液中提取出来;同时考察了波长和流动相配比对AMPS和AM分离效果的影响,得到了最佳色谱分析条件.实验结果表明,适宜的色谱分析条件为:采用ZORBAXSB-C18(4.0mm×50mm)液相色谱柱,以体积比为85∶15的甲醇-水混合液为流动相,流动相流1.0mL/min,检测波长220nm,柱温27℃,进样量6μL;建立了AMPS/AM二元共聚物中残余单体AMPS和AM的标准工作曲线,二者的线性相关系数均大于0.9995,回收率为97.50%~100.62%,相对标准偏差为0.4357%~1.2981%.该方法具有操作简便、分析速度快、分离效果好、灵敏、准确高等优点,可满足测试要求.

  3. Hydrosmotic effect of angiotensin II in the toad skin: role of cyclic AMP.

    Science.gov (United States)

    Coviello, A; Brauckmann, E S; de Atenor, M S; Apud, J A; Causarano, J

    1975-01-01

    The mechanism of action of the hydrosmotic response of the isolated skin of the toad Bufo arenarum Hensel to angiotensin II was studied by means of an indirect pharmacological approach. Angiotensin II (2.10(-10) M), vasopressin (2.10(-13) M) and theophylline (10(-4) and 10(-3) M) in subliminal doses produced a significant increase on water permeability when added in different paired combinations. Angiotensin II (2.10(-7) M) and vasopressin (2.10(-8) M) in doses producing significant effects on water permeability increased the response to submaximal doses of epinephrine (10(-6) M) but not to higher doses (10(-5) M). Acid pH (6.4) and prostaglandin E1 (2.10(-7) M) reduced significantly the hydrosmotic response to angiotensin II, but in contrast with the toad bladder, the effect was not completely abolished. Present results support the view that the hydrosmotic effect of angiotensin II in toad skin is mediated by the adenylate cyclase - cyclic AMP system. PMID:189568

  4. Transcriptional regulation induced by cAMP elevation in mouse Schwann cells

    Directory of Open Access Journals (Sweden)

    Daniela Schmid

    2014-04-01

    Full Text Available In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be modulated by forskolin. One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1, whose mRNA expression levels were reduced in treated Schwann cells. Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage. Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix, underlining its importance during Schwann cell differentiation in vitro. Comparison of samples derived from postnatal sciatic nerves and from both treated and untreated Schwann cell cultures showed considerable differences in gene expression between in vivo and in vitro, allowing us to separate Schwann cell autonomous from tissue-related changes. The whole data set of the cell culture microarray study is provided to offer an interactive search tool for genes of interest.

  5. Evolutionary paths of the cAMP-dependent protein kinase (PKA catalytic subunits.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available 3',5'-cyclic adenosine monophosphate (cAMP dependent protein kinase or protein kinase A (PKA has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods.

  6. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    Science.gov (United States)

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions. PMID:27357676

  7. Hypoxia regulates the cAMP- and Ca2+/calmodulin signaling systems in PC12 cells.

    Science.gov (United States)

    Beitner-Johnson, D; Leibold, J; Millhorn, D E

    1998-01-01

    Hypoxic/ischemic trauma is a primary factor in the pathology of various disease states. Yet, very little is known about the molecular mechanisms involved in cellular responses and adaptations to hypoxia. As a means of identifying intracellular signaling systems that are regulated in response to hypoxia, the effects of acute and chronic hypoxia on the activity of protein kinase A (PKA) and Ca2+/CaM-dependent protein kinase II (CaMK-II) were evaluated in rat pheochromocytoma (PC12) cells. Chronic (> 6 hr), but not acute exposure to hypoxia (5% O2) significantly decreased both PKA enzyme activity and immunoreactivity compared to control levels. This effect was not due to hypoxia-induced alterations in cell number or viability. Similarly, chronic hypoxia significantly decreased CaMK-II enzyme activity and protein levels in PC12 cells. These data demonstrate that down-regulation of the cAMP and Ca2+/CaM-signaling systems is a mechanism by which PC12 cells adapt to long-term hypoxia. PMID:9439610

  8. Spatial regulation of the cAMP-dependent protein kinase during chemotactic cell migration.

    Science.gov (United States)

    Howe, Alan K; Baldor, Linda C; Hogan, Brian P

    2005-10-01

    Historically, the cAMP-dependent protein kinase (PKA) has a paradoxical role in cell motility, having been shown to both facilitate and inhibit actin cytoskeletal dynamics and cell migration. In an effort to understand this dichotomy, we show here that PKA is regulated in subcellular space during cell migration. Immunofluorescence microscopy and biochemical enrichment of pseudopodia showed that type II regulatory subunits of PKA and PKA activity are enriched in protrusive cellular structures formed during chemotaxis. This enrichment correlates with increased phosphorylation of key cytoskeletal substrates for PKA, including the vasodilator-stimulated phosphoprotein (VASP) and the protein tyrosine phosphatase containing a PEST motif. Importantly, inhibition of PKA activity or its ability to interact with A kinase anchoring proteins inhibited the activity of the Rac GTPase within pseudopodia. This effect correlated with both decreased guanine nucleotide exchange factor activity and increased GTPase activating protein activity. Finally, inhibition of PKA anchoring, like inhibition of total PKA activity, inhibited pseudopod formation and chemotactic cell migration. These data demonstrate that spatial regulation of PKA via anchoring is an important facet of normal chemotactic cell movement.

  9. Stimulation of IGF-binding protein-1 secretion by AMP-activated protein kinase.

    Science.gov (United States)

    Lewitt, M S

    2001-04-20

    Insulin-like growth factor-binding protein-1 (IGFBP-1) is stimulated during intensive exercise and in catabolic conditions to very high concentrations, which are not completely explained by known regulators such as insulin and glucocorticoids. The role of AMP-activated protein kinase (AMPK), an important signaling system in lipid and carbohydrate metabolism, in regulating IGFBP-1 was studied in H4-II-E rat hepatoma cells. Arsenic(III) oxide and 5-aminoimidazole-4-carboxamide-riboside (AICAR) were used as activators. AICAR (150 microM) stimulated IGFBP-1 secretion twofold during a 5-h incubation (P = 0.002). Insulin (100 ng/ml) inhibited IGFBP-1 by 80% (P < 0.001), but this was completely abolished in the presence of 150 microM AICAR. The effect of dexamethasone in stimulating IGFBP-1 threefold was additive to the effect of AICAR (P < 0.001) and, in the presence of AICAR, was incompletely inhibited by insulin. In conclusion AMPK is identified as a novel regulatory pathway for IGFBP-1, stimulating secretion and blocking the inhibitory effect of insulin. PMID:11302732

  10. AMP-activated protein kinase is activated by non-steroidal anti-inflammatory drugs.

    Science.gov (United States)

    King, Tanya S; Russe, Otto Quintus; Möser, Christine V; Ferreirós, Nerea; Kynast, Katharina L; Knothe, Claudia; Olbrich, Katrin; Geisslinger, Gerd; Niederberger, Ellen

    2015-09-01

    AMP-activated kinase (AMPK) is a cellular energy sensor, which is activated in stages of increased adenosine triphosphate (ATP) consumption. Its activation has been associated with a number of beneficial effects such as decrease of inflammatory processes and inhibition of disease progression of diabetes and obesity. A recent study suggested that salicylate, the active metabolite of the non-steroidal anti-inflammatory drug (NSAID) acetyl-salicylic acid (aspirin), is able to activate AMPK pharmacologically. This observation raised the question whether or not other NSAIDs might also act as AMPK activators and whether this action might contribute to their cyclooxygenase (COX)-independent anti-inflammatory properties. In this study, we investigated mouse and human neuronal cells and liver tissue of mice after treatment with various NSAIDs. Our results showed that the non-selective acidic NSAIDs ibuprofen and diclofenac induced AMPK activation similar to aspirin while the COX-2 selective drug etoricoxib and the non-opioid analgesic paracetamol, both drugs have no acidic structure, failed to activate AMPK. In conclusion, our results revealed that AMPK can be activated by specific non-steroidal anti-inflammatory drugs such as salicylic acid, ibuprofen or diclofenac possibly depending on the acidic structure of the drugs. AMPK might therefore contribute to their antinociceptive and anti-inflammatory properties. PMID:26049010

  11. Purification and Characterization of Cyclic AMP-Binding Protein from Ganoderma lucidum

    Institute of Scientific and Technical Information of China (English)

    WANG Qi; KIM Jung-Sik; CHUNG Ki-Chul

    2004-01-01

    Cyclic AMP-binding protein was purified 30 fold from the mycelia of Ganoderma lucidum by the methods of ammonium sulfate precipitation, DEAE-cellulose, phospho-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. The molecular mass of the purified protein is 34.5 kDa and 17 kDa by Sephacryl S-100 gel filtration and SDS-ployacrylamide gel electrophoresis, respectively. From these results it is suggested that the protein has a homometric dimmer structure. The pI of the purified protein is pH 8. 2 by native isoelectric focusing gel. The half-life of the protein activity in 10% glycerol at 4 ℃ is 7 d in crude extract, but its half-life is only 3 d under purifying conditions. The optimal conditions of the protein activity are at 1 ℃ and pH 7. 5. Its activity is increased 6 times by 1 mmol/L Zn2+ and is slightly inhibited by cGMP,Cu2+ and Mn2+.

  12. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP).

    Science.gov (United States)

    Hallberg, Zachary F; Wang, Xin C; Wright, Todd A; Nan, Beiyan; Ad, Omer; Yeo, Jongchan; Hammond, Ming C

    2016-02-16

    Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

  13. Danthron activates AMP-activated protein kinase and regulates lipid and glucose metabolism in vitro

    Institute of Scientific and Technical Information of China (English)

    Rong ZHOU; Ling WANG; Xing XU; Jing CHEN; Li-hong HU; Li-li CHEN; Xu SHEN

    2013-01-01

    Aim:To discover the active compound on AMP-activated protein kinase (AMPK) activation and investigate the effects of the active compound 1,8-dihydroxyanthraquinone (danthron) from the traditional Chinese medicine rhubarb on AMPK-mediated lipid and glucose metabolism in vitro.Methods:HepG2 and C2C12 cells were used.Cell viability was determined using MTT assay.Real-time PCR was performed to measure the gene expression.Western blotting assay was applied to investigate the protein phosphorylation level.Enzymatic assay kits were used to detect the total cholesterol (TC),triglyceride (TG) and glucose contents.Results:Danthron (0.1,1,and 10 μmol/L) dose-dependently promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC)in both HepG2 and C2C12 cells.Meanwhile,danthron treatment significantly reduced the lipid synthesis related sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthetase (FAS) gene expressions,and the TC and TG levels.In addition,danthron treatment efficiently increased glucose consumption.The actions of danthron on lipid and glucose metabolism were abolished or reversed by co-treatment with the AMPK inhibitor compound C.Conclusion:Danthron effectively reduces intracellular lipid contents and enhanced glucose consumption in vitro via activation of AMPK signaling pathway.

  14. Curcumin attenuates diet-induced hepatic steatosis by activating AMP-activated protein kinase.

    Science.gov (United States)

    Um, Min Young; Hwang, Kwang Hyun; Ahn, Jiyun; Ha, Tae Youl

    2013-09-01

    Curcumin is a well-known component of traditional turmeric (Curcuma longa), which has been reported to prevent obesity and diabetes. However, the effect of curcumin on hepatic lipid metabolism remains unclear. The aim of this study was to examine the effects of curcumin on hepatic steatosis in high-fat/cholesterol diet (HFD)-induced obese mice. Male C57BL/6J mice were fed a normal diet (ND), HFD or HFD with 0.15% curcumin (HFD+C) for 11 weeks. We found that curcumin significantly lowered the body-weight and adipose tissue weight of mice in the HFD+C group compared with the findings for the HFD group (p cholesterol, fasting glucose and insulin in serum were decreased, and HFD-induced impairment of insulin sensitivity was improved by curcumin supplementation (p Curcumin protected against the development of hepatic steatosis by reducing hepatic fat accumulation. Moreover, curcumin activated AMP-activated protein kinase (AMPK) and elevated the gene expression of peroxisome proliferator-activated receptor alpha. By contrast, curcumin suppressed the HFD-mediated increases in sterol regulatory element-binding protein-1, acetyl-CoA carboxylase 1, fatty acid synthase and cluster of differentiation 36 expression. Taken together, these findings indicate that curcumin attenuates HFD-induced hepatic steatosis by regulating hepatic lipid metabolism via AMPK activation, suggesting its use as a therapeutic for hepatic steatosis.

  15. 5-ALA mediated photodynamic therapy induces autophagic cell death via AMP-activated protein kinase

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    Lin Yu-Hsin

    2010-04-01

    Full Text Available Abstract Photodynamic therapy (PDT has been developed as an anticancer treatment, which is based on the tumor-specific accumulation of a photosensitizer that induces cell death after irradiation of light with a specific wavelength. Depending on the subcellular localization of the photosensitizer, PDT could trigger various signal transduction cascades and induce cell death such as apoptosis, autophagy, and necrosis. In this study, we report that both AMP-activated protein kinase (AMPK and mitogen-activated protein kinase (MAPK signaling cascades are activated following 5-aminolevulinic acid (ALA-mediated PDT in both PC12 and CL1-0 cells. Although the activities of caspase-9 and -3 are elevated, the caspase inhibitor zVAD-fmk did not protect cells against ALA-PDT-induced cell death. Instead, autophagic cell death was found in PC12 and CL1-0 cells treated with ALA-PDT. Most importantly, we report here for the first time that it is the activation of AMPK, but not MAPKs that plays a crucial role in mediating autophagic cell death induced by ALA-PDT. This novel observation indicates that the AMPK pathway play an important role in ALA-PDT-induced autophagy.

  16. Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding.

    Science.gov (United States)

    Kuznetsov, Aleksei; Kivi, Rait; Järv, Jaak

    2016-04-01

    Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein. PMID:26896699

  17. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    Directory of Open Access Journals (Sweden)

    Maria Ranieri-Raggi

    2014-05-01

    Full Text Available Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD and the metal binding protein histidine-proline-rich glycoprotein (HPRG acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua(μ-carboxylatodizinc(II core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated.

  18. A novel indirect sequence readout component in the E. coli cyclic AMP receptor protein operator.

    Science.gov (United States)

    Lindemose, Søren; Nielsen, Peter Eigil; Valentin-Hansen, Poul; Møllegaard, Niels Erik

    2014-03-21

    The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO2(2+)) probing data, we propose that the underlying mechanism relies on N6 deformability.

  19. cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

    Energy Technology Data Exchange (ETDEWEB)

    Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui; Ha, Hyunil, E-mail: hyunil74@hotmail.com; Lee, Zang Hee, E-mail: zang1959@snu.ac.kr

    2010-07-23

    Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

  20. Metabolic Basis for Thyroid Hormone Liver Preconditioning: Upregulation of AMP-Activated Protein Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Luis A. Videla

    2012-01-01

    Full Text Available The liver is a major organ responsible for most functions of cellular metabolism and a mediator between dietary and endogenous sources of energy for extrahepatic tissues. In this context, adenosine-monophosphate- (AMP- activated protein kinase (AMPK constitutes an intrahepatic energy sensor regulating physiological energy dynamics by limiting anabolism and stimulating catabolism, thus increasing ATP availability. This is achieved by mechanisms involving direct allosteric activation and reversible phosphorylation of AMPK, in response to signals such as energy status, serum insulin/glucagon ratio, nutritional stresses, pharmacological and natural compounds, and oxidative stress status. Reactive oxygen species (ROS lead to cellular AMPK activation and downstream signaling under several experimental conditions. Thyroid hormone (L-3,3′,5-triiodothyronine, T3 administration, a condition that enhances liver ROS generation, triggers the redox upregulation of cytoprotective proteins affording preconditioning against ischemia-reperfusion (IR liver injury. Data discussed in this work suggest that T3-induced liver activation of AMPK may be of importance in the promotion of metabolic processes favouring energy supply for the induction and operation of preconditioning mechanisms. These include antioxidant, antiapoptotic, and anti-inflammatory mechanisms, repair or resynthesis of altered biomolecules, induction of the homeostatic acute-phase response, and stimulation of liver cell proliferation, which are required to cope with the damaging processes set in by IR.

  1. Jellyfish vision starts with cAMP signaling mediated by opsin-G(s) cascade.

    Science.gov (United States)

    Koyanagi, Mitsumasa; Takano, Kosuke; Tsukamoto, Hisao; Ohtsu, Kohzoh; Tokunaga, Fumio; Terakita, Akihisa

    2008-10-01

    Light sensing starts with phototransduction in photoreceptor cells. The phototransduction cascade has diverged in different species, such as those mediated by transducin in vertebrate rods and cones, by G(q)-type G protein in insect and molluscan rhabdomeric-type visual cells and vertebrate photosensitive retinal ganglion cells, and by G(o)-type G protein in scallop ciliary-type visual cells. Here, we investigated the phototransduction cascade of a prebilaterian box jellyfish, the most basal animal having eyes containing lens and ciliary-type visual cells similar to vertebrate eyes, to examine the similarity at the molecular level and to obtain an implication of the origin of the vertebrate phototransduction cascade. We showed that the opsin-based pigment functions as a green-sensitive visual pigment and triggers the G(s)-type G protein-mediated phototransduction cascade in the ciliary-type visual cells of the box jellyfish lens eyes. We also demonstrated the light-dependent cAMP increase in the jellyfish visual cells and HEK293S cells expressing the jellyfish opsin. The first identified prebilaterian cascade was distinct from known phototransduction cascades but exhibited significant partial similarity with those in vertebrate and molluscan ciliary-type visual cells, because all involved cyclic nucleotide signaling. These similarities imply a monophyletic origin of ciliary phototransduction cascades distributed from prebilaterian to vertebrate. PMID:18832159

  2. Resveratrol improves non-alcoholic fatty liver disease by activating AMP-activated protein kinase

    Institute of Scientific and Technical Information of China (English)

    Jing SHANG; Lu-lu CHEN; Eang-xi XIAO; Hui SUN; Hong-cheng DING; Hu XIAO

    2008-01-01

    Aim: To investigate whether resveratrol (RSV) can improve non-alcoholic fatty liver disease (NAFLD) and to find the possible mechanism. Methods: Rats fed a high-fat diet were treated with RSV. The liver histology was observed. Hyperinsulinemic euglycemic clamp was performed to assess insulin sensitivity. Fat accumulation was induced in HepG2 cells, and the cells were treated with RSV. AMP-activated protein kinase (AMPK) phosphorylation levels were de-termined both in the animal study and cell study. Results: Rats fed a high-fat diet developed abdominal obesity, NAFLD, and insulin resistance (IR), which were markedly improved by 10 weeks of RSV administration. RSV treatment prevented triacylglycerol (TG) accumulation in HepG2 cells that were incubated with high concentration of glucose and insulin. Both in vivo and in vitro studies showed that RSV treatment could promote the phosphorylation of AMPK, which in this study, suppressed 2 lipogenesis gene expressions, contributing to the improvement of NAFLD and IR. Conclusion: The results indicated that by re-ducing TG accumulation and improving IR, RSV could protect the liver from NAFLD. The activation of AMPK was involved in the mechanism. RSV has the therapeutic potential for preventing or treating NAFLD and IR-related metabolic disorders.

  3. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    Science.gov (United States)

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

  4. Plasmid-mediated AmpC: prevalence in community-acquired isolates in Amsterdam, the Netherlands, and risk factors for carriage.

    Directory of Open Access Journals (Sweden)

    E Ascelijn Reuland

    Full Text Available The objective of this study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains.Fecal samples were obtained from community-dwelling volunteers. Participants also returned a questionnaire for analysis of risk factors. Screening for pAmpC was performed with selective enrichment broth and a selective screening agar. Confirmation of AmpC-production was performed with two double disc combination tests: cefotaxime and ceftazidime with either boronic acid or cloxacillin as inhibitor. Multiplex PCR was used as gold standard for detection of pAmpC. 16S rRNA PCR and AFLP were performed as required, plasmids were identified by PCR-based replicon typing. Questionnaire results were analyzed with SPSS, version 20.0.Fecal samples were obtained from 550 volunteers; mean age 51 years (range: 18-91, 61% were females. pAmpC was present in seven E. coli isolates (7/550, 1.3%, 0.6-2.7 95% CI: six CMY-2-like pAmpC and one DHA. ESBL-encoding genes were found in 52/550 (9.5%, 7.3-12.2 95% CI isolates; these were predominantly blaCTX-M genes. Two isolates had both ESBL and pAmpC. Admission to a hospital in the previous year was the only risk factor we identified.Our data indicate that the prevalence of pAmpC in the community seems still low. However, since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, there is consistent risk that further increase of their prevalence might go undetected.

  5. Activation of exchange protein activated by cAMP in the rat basolateral amygdala impairs reconsolidation of a memory associated with self-administered cocaine.

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    Xun Wan

    Full Text Available The intracellular mechanisms underlying memory reconsolidation critically involve cAMP signaling. These events were originally attributed to PKA activation by cAMP, but the identification of Exchange Protein Activated by cAMP (Epac, as a distinct mediator of cAMP signaling, suggests that cAMP-regulated processes that subserve memory reconsolidation are more complex. Here we investigated how activation of Epac with 8-pCPT-cAMP (8-CPT impacts reconsolidation of a memory that had been associated with cocaine self-administration. Rats were trained to lever press for cocaine on an FR-1 schedule, in which each cocaine delivery was paired with a tone+light cue. Lever pressing was then extinguished in the absence of cue presentations and cocaine delivery. Following the last day of extinction, rats were put in a novel context, in which the conditioned cue was presented to reactivate the cocaine-associated memory. Immediate bilateral infusions of 8-CPT into the basolateral amygdala (BLA following reactivation disrupted subsequent cue-induced reinstatement in a dose-dependent manner, and modestly reduced responding for conditioned reinforcement. When 8-CPT infusions were delayed for 3 hours after the cue reactivation session or were given after a cue extinction session, no effect on cue-induced reinstatement was observed. Co-administration of 8-CPT and the PKA activator 6-Bnz-cAMP (10 nmol/side rescued memory reconsolidation while 6-Bnz alone had no effect, suggesting an antagonizing interaction between the two cAMP signaling substrates. Taken together, these studies suggest that activation of Epac represents a parallel cAMP-dependent pathway that can inhibit reconsolidation of cocaine-cue memories and reduce the ability of the cue to produce reinstatement of cocaine-seeking behavior.

  6. 高效液相色谱法分离纯化小皱蝽抗菌肽AMP-1%PURIFICATION OF THE ANTIMICROBIAL PEPTIDE AMP- 1 OF CYCLOPELTA PARVA DISTANT BY HPIC

    Institute of Scientific and Technical Information of China (English)

    杨泽宏; 杨星勇; 袁小红; 罗克明; 罗小英; 张永军; 郭余龙; 裴炎

    2001-01-01

    An antimicrobial peptide, AMP- 1,was isolated from the bug Cyclopelta parva Distant,an insect of Pentatomidae, Hemiptera, by the extraction and preparation of the crude extract with the acid extraction methods and fractionation of Sep- Pak C1s Cartridge. Subsequently,40% Sep - Pak fraction was purified by the initial reverse phase HPLC. The active peak was further fractionated by gel - permeation chromatography HPLC and the final reverse phase HPLC. All the HPLC purifications utilized the Acetonitrile - Water- Trifluoroacetic acetic elution system as mobile phase, with detection at 225 nm. The pure substances were recovered and the anti - bacterial activity of each aliquot was routinely monitored by the plate growth inhinhibition assay on S. aureus and E. coli, AMP- 1 is the antimicrobial polypeptide.%用昆虫生理盐水浸提小皱蝽成虫,浸提液冻干后,酸提取法制备粗提液,固相萃取(SPE)初分级分离,活性洗脱组分反相高效液相色谱(RP-HPKLC)纯化,进一步凝胶渗透色谱(GPC)及反相高效液相色谱(RP-HPLC)纯化,分离得到一抗菌肽,茚三酮反应黄色,命名为AMP-1。在190nm~225nm短波紫外区,AMP-1有紫外吸收。

  7. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

    Directory of Open Access Journals (Sweden)

    Philip D Townsend

    Full Text Available The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  8. Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

    OpenAIRE

    Motawea, Hanaa K. B.; Selvi C. Jeyaraj; Eid, Ali H; Mitra, Srabani; Unger, Nicholas T.; Ahmed, Amany A. E.; Flavahan, Nicholas A.; Chotani, Maqsood A.

    2013-01-01

    The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expressio...

  9. Enhancement of Inhibitory Avoidance and Conditioned Taste Aversion Memory With Insular Cortex Infusions of 8-Br-cAMP: Involvement of the Basolateral Amygdala

    OpenAIRE

    Miranda, María I.; McGaugh, James L.

    2004-01-01

    There is considerable evidence that in rats, the insular cortex (IC) and amygdala are involved in the learning and memory of aversively motivated tasks. The present experiments examined the effects of 8-Br-cAMP, an analog of cAMP, and oxotremorine, a muscarinic agonist, infused into the IC after inhibitory avoidance (IA) training and during the acquisition/consolidation of conditioned taste aversion (CTA). Posttraining infusion into the IC of 0.3 μg oxotremorine and 1.25 μg 8-Br-cAMP enhanced...

  10. Listeria monocytogenes Multidrug Resistance Transporters and Cyclic Di-AMP, Which Contribute to Type I Interferon Induction, Play a Role in Cell Wall Stress

    OpenAIRE

    Kaplan Zeevi, Millie; Shafir, Nirit S.; Shaham, Shira; Friedman, Sivan; Sigal, Nadejda; Nir Paz, Ran; Boneca, Ivo G; Herskovits, Anat A.

    2013-01-01

    The intracellular bacterial pathogen Listeria monocytogenes activates a robust type I interferon response upon infection. This response is partially dependent on the multidrug resistance (MDR) transporter MdrM and relies on cyclic-di-AMP (c-di-AMP) secretion, yet the functions of MdrM and cyclic-di-AMP that lead to this response are unknown. Here we report that it is not MdrM alone but a cohort of MDR transporters that together contribute to type I interferon induction during infection. In a ...

  11. Reciprocal bystander effect between α-irradiated macrophage and hepatocyte is mediated by cAMP through a membrane signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    He, Mingyuan [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China); Department of Radiation Oncology, China–Japan Union Hospital of Jilin University, Changchun 130033 (China); Dong, Chen; Xie, Yuexia; Li, Jitao; Yuan, Dexiao; Bai, Yang [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China); Shao, Chunlin, E-mail: clshao@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, No. 2094 Xie-Tu Road, Shanghai 200032 (China)

    2014-05-15

    Highlights: • α-Irradiation induced reciprocal effects between macrophage and hepatocyte cells. • cAMP played a protective role in regulating the reverse bystander effect. • cAMP communication contributed to the reciprocal effects via membrane signaling. • p53 was required for cAMP-regulated bystander effect in the recipient cells. - Abstract: Irradiated cells can induce biological effects on vicinal non-irradiated bystander cells, meanwhile the bystander cells may rescue the irradiated cells through a feedback signal stress. To elucidate the nature of this reciprocal effect, we examined the interaction between α-irradiated human macrophage cells U937 and its bystander HL-7702 hepatocyte cells using a cell co-culture system. Results showed that after 6 h of cell co-culture, mitochondria depolarization corresponding to apoptosis was significantly induced in the HL-7702 cells, but the formation of micronuclei in the irradiated U937 cells was markedly decreased compared to that without cell co-culture treatment. This reciprocal effect was not observed when the cell membrane signaling pathway was blocked by filipin that inhibited cAMP transmission from bystander cells to irradiated cells. After treatment of cells with exogenous cAMP, forskolin (an activator of cAMP) or KH-7 (an inhibitor of cAMP), respectively, it was confirmed that cAMP communication from bystander cells to targeted cells could mitigate radiation damage in U739 cells, and this cAMP insufficiency in the bystander cells contributed to the enhancement of bystander apoptosis. Moreover, the bystander apoptosis in HL-7702 cells was aggravated by cAMP inhibition but it could not be evoked when p53 of HL-7702 cells was knocked down no matter of forskolin and KH-7 treatment. In conclusion, this study disclosed that cAMP could be released from bystander HL-7702 cells and compensated to α-irradiated U937 cells through a membrane signaling pathway and this cAMP communication played a profound role in

  12. A positive feedback loop of phosphodiesterase 3 (PDE3) and inducible cAMP early repressor (ICER) leads to cardiomyocyte apoptosis

    OpenAIRE

    Ding, Bo; Abe, Jun-ichi; Wei, Heng; Xu, Haodong; Che, Wenyi; Aizawa, Toru; Liu, Weimin; Molina, Carlos A.; Sadoshima, Junichi; Blaxall, Burns C.; Berk, Bradford C.; Yan, Chen

    2005-01-01

    cAMP plays crucial roles in cardiac remodeling and the progression of heart failure. Recently, we found that expression of cAMP hydrolyzing phosphodiesterase 3A (PDE3A) was significantly reduced in human failing hearts, accompanied by up-regulation of inducible cAMP early repressor (ICER) expression. Angiotensin II (Ang II) and the β-adrenergic receptor agonist isoproterenol (ISO) also induced persistent PDE3A down-regulation and concomitant ICER up-regulation in vitro, which is important in ...

  13. Serosal Zn2+ inhibits 8-Br-cAMP stimulated chloride secretion in piglet small intestinal epithelium in vitro

    DEFF Research Database (Denmark)

    Carlson, Dorthe; Sehested, Jakob; Poulsen, Hanne Damgaard

    2010-01-01

    . Piglets (n = 24) were weaned at 28 days of age and allocated at two dietary treatments (ZnO0 and ZnO2500) and at 5-6 days after weaning the piglets were slaughtered and small intestinal epithelium from each piglet was mounted into 8 Ussing chambers. The effect of 23 μM serosal Zn2+ on 8-Br-cAMP (8...... improved piglet performance and zinc status, but did not affect the in vitro results. Serosal Zn2+ reduced 8-Br-cAMP and 5-HT induced secretion. But FSK induced secretion was only reduced by serosal Zn2+ when the K+ channel blocker was present indicating interactions between 293B and Zn2+. These data...

  14. Accurate convergent finite difference approximations for viscosity solutions of the elliptic Monge-Amp\\`ere partial differential equation

    CERN Document Server

    Froese, Brittany D

    2012-01-01

    The theory of viscosity solutions has been effective for representing and approximating weak solutions to fully nonlinear Partial Differential Equations (PDEs) such as the elliptic Monge-Amp\\`ere equation. The approximation theory of Barles-Souganidis [Barles and Souganidis, Asymptotic Anal., 4 (1999) 271-283] requires that numerical schemes be monotone (or elliptic in the sense of [Oberman, SIAM J. Numer. Anal, 44 (2006) 879-895]. But such schemes have limited accuracy. In this article, we establish a convergence result for nearly monotone schemes. This allows us to construct finite difference discretizations of arbitrarily high-order. We demonstrate that the higher accuracy is achieved when solutions are sufficiently smooth. In addition, the filtered scheme provides a natural detection principle for singularities. We employ this framework to construct a formally second-order scheme for the Monge-Amp\\`ere equation and present computational results on smooth and singular solutions.

  15. Non-coding RNA LINC00473 mediates decidualization of human endometrial stromal cells in response to cAMP signaling.

    Science.gov (United States)

    Liang, Xiao-Huan; Deng, Wen-Bo; Liu, Yue-Fang; Liang, Yu-Xiang; Fan, Zong-Min; Gu, Xiao-Wei; Liu, Ji-Long; Sha, Ai-Guo; Diao, Hong-Lu; Yang, Zeng-Ming

    2016-01-01

    Decidualization is an essential step in the establishment of pregnancy. However, the functional contributions of long intergenic noncoding RNAs (LincRNAs) to decidualization have not been explored. To explore the regulation and role of LincRNAs during human decidualization, human endometrial stromal cells (HESCs) are induced to undergo in vitro decidualization by treating with estradiol-17β, db-cAMP and medroxyprogesterone acetate. LINC00473 (LINC473) expression is highly induced in HESCs after decidual stimulus. We found that cAMP-PKA pathway regulates the expression of LINC473 through IL-11-mediated STAT3 phosphorylation. RNA interference-mediated down-regulation of LINC473 inhibits in vitro decidualization. These results suggested that LINC473 might be functionally required for human decidualization. This is the first report demonstrating the presence of LincRNA during human decidualization. PMID:26947914

  16. Non-coding RNA LINC00473 mediates decidualization of human endometrial stromal cells in response to cAMP signaling

    Science.gov (United States)

    Liang, Xiao-Huan; Deng, Wen-Bo; Liu, Yue-Fang; Liang, Yu-Xiang; Fan, Zong-Min; Gu, Xiao-Wei; Liu, Ji-Long; Sha, Ai-Guo; Diao, Hong-Lu; Yang, Zeng-Ming

    2016-01-01

    Decidualization is an essential step in the establishment of pregnancy. However, the functional contributions of long intergenic noncoding RNAs (LincRNAs) to decidualization have not been explored. To explore the regulation and role of LincRNAs during human decidualization, human endometrial stromal cells (HESCs) are induced to undergo in vitro decidualization by treating with estradiol-17β, db-cAMP and medroxyprogesterone acetate. LINC00473 (LINC473) expression is highly induced in HESCs after decidual stimulus. We found that cAMP-PKA pathway regulates the expression of LINC473 through IL-11-mediated STAT3 phosphorylation. RNA interference-mediated down-regulation of LINC473 inhibits in vitro decidualization. These results suggested that LINC473 might be functionally required for human decidualization. This is the first report demonstrating the presence of LincRNA during human decidualization. PMID:26947914

  17. CARDIAC RISK STRATIFICATION IN PATIENTS WITH CONGESTIVE HEART FAILURE: A CATECHOLAMINES-β- ADRENOCEPTOR-cAMP PATHWAY

    Institute of Scientific and Technical Information of China (English)

    Ying-xin Peng; Jiang Shan; Su-jun Zhang; Chun-li Rong; Jun-ping Li; Na Wang; Hao Xue; Shi-ling Zheng; Min Wu

    2005-01-01

    Objective To investigate the stratification risk of catecholamines-β-adrenoceptor (β-AR)-cAMP pathway for cardiogenic death events in patients with congestive heart failure (CHF).Methods A total of 83 identified CHF patients with a baseline and follow-up plasma levels of norepinephrine (NE)and epinephrine (E), lymphocytes β-AR density (Bmax), and intralymphocyte cAMP content in peripheral blood were followed up. Major cardiogenic death events were registered.Results The period between the initial entry and the last follow-up measurement were 51± 16 months, the total duration of clinical follow-up after the last measurement were 14±8 months. During follow-up, 39 patients died of cardiogenic (sudden death 17 patients, worsening heart failure 22 patients). Persistence of high NE, E, and cAMP from baseline to follow-up were confirmed as risk predicting factors of cardiovascular events. Persistence NE above 4.0 nmol/L, E above adverse prognostic predictors. The major cardiogenic death events rates per 100 patients-years were 1.33 and 4.82 in patients with NE below and above 4.0 nmol/L (HR: 2.91; 95% CI: 1.08-7.33; P = 0.015); were 1.42 and 4.36 in the patients with E levels below and above 3.5 nmol/L (HR: 2.64; 95% CI: 1.02-6.41; P = 0.019); were 1.81 and 4.67 in the 0.017), but difference was not significant between the β-AR density below and above median.Conclusions Persistent increase in circulating catecholamines and intralymphocyte cAMP content may increase the long-term mortality in CHF patients.

  18. Optimizing the Emitter Layer for Higher Efficiency Solar Cell Based SiGe Using AMPS1D

    OpenAIRE

    Boukais Meriem; B. Dennai; A. Ould-Abbas

    2015-01-01

    The thin-film SiGe is considered as promising candidate to meet the outstanding need for photovoltaic applications with enhanced adsorption characteristics and improved conversion efficiency [1-6]. In this paper, we simulated a solar cell type SiGe using AMPS1D (Analysis of Microelectronic and photonic structure) developed at Pennsylvania State University, to analyze emitter layer (thickness, doping) and we studied their influence on the photovoltaic solar cell. The simulation result shows th...

  19. Simulation of Hetero-junction (GaInP/GaAs Solar Cell Using AMPS-1D

    Directory of Open Access Journals (Sweden)

    Dennai Benmoussa

    2016-03-01

    Full Text Available Photovoltaic conversion is the direct conversion of electromagnetic energy into electrical energy continuously. This electromagnetic energy is the most solar radiation. In this work we performed a computer modelling using AMPS 1D optimization of hetero-junction solar cells GaInP / GaAs configuration for p/n. We studied the influence of the thickness the base layer in the cell offers on the open circuit voltage, the short circuit current and efficiency.

  20. Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway.

    Directory of Open Access Journals (Sweden)

    Aishe Chen

    Full Text Available Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2',5'-dideoxyadenosine (DDA or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a G(i signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the G(s/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the G(s/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells.

  1. Validation of Testing and Interpretation Protocols for Low Template DNA Samples Using AmpFℓSTR® Identifiler®

    OpenAIRE

    Caragine, Theresa; Mikulasovich, Rebecca; Tamariz, Jeannie; Bajda, Ewelina; Sebestyen, James; Baum, Howard; Prinz, Mechthild

    2009-01-01

    Aim To test the reliability, robustness, and reproducibility of short tandem repeat (STR) profiling of low template DNA (LT-DNA) when employing a defined set of testing and interpretation parameters. Methods DNA from known donors was measured with a quantitative real time polymerase chain reaction (PCR) assay that consistently detects less than 1 pg/μL of DNA within a factor of 0.3. Extracts were amplified in triplicate with AmpFSTR® Identifiler® reagents under enhanc...

  2. Adiponectin Stimulates Angiogenesis by Promoting Cross-talk between AMP-activated Protein Kinase and Akt Signaling in Endothelial Cells*

    OpenAIRE

    Ouchi, Noriyuki; Kobayashi, Hideki; Kihara, Shinji; Kumada, Masahiro; Sato, Kaori; Inoue, Tatsuya; Funahashi, Tohru; Walsh, Kenneth

    2003-01-01

    Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein ...

  3. Simulation of Hetero-junction (GaInP/GaAs) Solar Cell Using AMPS-1D

    OpenAIRE

    Dennai Benmoussa; M. Boukais; H. Benslimane

    2016-01-01

    Photovoltaic conversion is the direct conversion of electromagnetic energy into electrical energy continuously. This electromagnetic energy is the most solar radiation. In this work we performed a computer modelling using AMPS 1D optimization of hetero-junction solar cells GaInP / GaAs configuration for p/n. We studied the influence of the thickness the base layer in the cell offers on the open circuit voltage, the short circuit current and efficiency.

  4. Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo*

    OpenAIRE

    Lee-Young, Robert S; Griffee, Susan R.; Lynes, Sara E.; Bracy, Deanna P.; Julio E Ayala; McGuinness, Owen P.; Wasserman, David H.

    2009-01-01

    AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, w...

  5. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    Yamauchi Mika

    2007-11-01

    Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

  6. Both cyclic-AMP and cyclic-GMP can act as regulators of the phenylpropanoid pathway in Arabidopsis thaliana seedlings.

    Science.gov (United States)

    Pietrowska-Borek, Małgorzata; Nuc, Katarzyna

    2013-09-01

    Cyclic nucleotides (cAMP and cGMP) are important signaling molecules that control a range of cellular functions and modulate different reactions. It is known that under abiotic or biotic stress plant cells synthesize these nucleotides and that they also enhance the activity of the phenylpropanoid pathway. Wondering what is the relation between these two facts, we investigated how the exogenously applied membrane-permeable derivatives, 8-Br-cAMP or 8-Br-cGMP, which are believed to act as the original cyclic nucleotides, affect the expression of the genes for and the specific activity of three enzymes of the phenylpropanoid pathway in Arabidopsis thaliana seedlings. We found that the expression of the genes of phenylalanine ammonia-lyase (PAL2), 4-coumarate:coenzyme A ligase (4CL1) and chalcone synthase (CHS), and the specific activities of PAL (EC 4.3.1.5), 4CL (EC 6.2.1.12) and CHS (EC 2.3.1.74) were induced in the same way by either of these cyclic nucleotides used at 5 μM concentration. None of the possible cAMP and cGMP degradation products (AMP, GMP, adenosine or guanosine) evoked such effects. Expression of PAL1, 4CL2 and 4CL3 were practically not affected. Although the investigated nucleotides induced rapid expression of the aforementioned enzymes, they did not affect the level of anthocyanins within the same period. We discuss the effects exerted by the exogenously administered cyclic nucleotides, their relation with stress and the role which the phenylpropanoid pathways the cyclic nucleotides may play in plants.

  7. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-12-01

    The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

  8. Expression and beta-glucan binding properties of Scots pine (Pinus sylvestris L.) antimicrobial protein (Sp-AMP)

    OpenAIRE

    Sooriyaarachchi, Sanjeewani; Jaber, Emad; Covarrubias, Adrian Suarez; Ubhayasekera, Wimal; Asiegbu, Frederick O.; Mowbray, Sherry L.

    2011-01-01

    Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host tree...

  9. A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction

    OpenAIRE

    Fusco, Salvatore; Ripoli, Cristian; Podda, Maria Vittoria; Ranieri, Sofia Chiatamone; Leone, Lucia; Toietta, Gabriele; McBurney, Michael W.; Schütz, Günther; Riccio, Antonella; Grassi, Claudio; Galeotti, Tommaso; Pani, Giovambattista

    2011-01-01

    Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD+-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes r...

  10. Localization of the catalytic subunit of cyclic AMP-dependent. Protein kinase in cultured cells using a specific antibody

    OpenAIRE

    1982-01-01

    We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C- subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit- Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoe...

  11. Influence on Ratio of AMP/ATP and AMPK Activity in Rat's Skeletal Muscle by Different Exercise Intensity%不同强度运动对大鼠骨骼肌AMP/ATP比值和AMPK活性的影响

    Institute of Scientific and Technical Information of China (English)

    张国华; 朱一力; 曾凡星

    2008-01-01

    目的:研究不同强度运动大鼠腓肠肌AMP、ATP含量及AMPK活性,旨在阐明不同强度运动时AMP/ATP的比值和AMPK的变化特点及二者的关系.方法:将62只雄性SD大鼠分为4大组:安静对照组、小强度运动组、中强度运动组、大强度运动组,其中运动组又各分为3小组,分别在运动后即刻、1 h和6 h取材.小、中和大强度一次性跑台运动的速度分别为10 m/min、18 m/min、26 m/min,坡度10°,时间60 min.AMP、ATP含量测定采用高效液相色谱法(HPLC),AMPK活性测定采用放射性同位素法.结果:小强度运动后AMPK活性不变,中到大强度运动后即刻分别增加50%(P<0.01)和1.8倍(P<0.01)并持续至1h,6 h回到安静水平;中到大强度即刻AMP分别增加16%(P<0.05)和62%(P<0.01),AMP/ATP分别升高33%(P<0.05)和89%(P<0.01),均在1 h回到安静水平;不同强度运动后即刻AMPK与AMP/ATP呈显著正相关(r=0.89).结论:1)AMPK在小强度运动时未激活,中到大强度运动时呈强度依赖性升高;2)运动激活AMPK的机制主要受AMP/ATP比值升高调控,而比值的升高主要依赖于AMP的增加而不是ATP的减少;3)运动后AMPK活性的恢复滞后于AMP/ATP比值的恢复.

  12. Transcutaneous electrical nerve stimulation (TENS) improves the diabetic cytopathy (DCP) via up-regulation of CGRP and cAMP.

    Science.gov (United States)

    Ding, Liucheng; Song, Tao; Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n=15), DM group (n=15) and control group (n=15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG. PMID:23468996

  13. NTP pattern of avian embryonic red cells: role of RNA degradation and AMP deaminase/5'-nucleotidase activity.

    Science.gov (United States)

    Baumann, Rosemarie; Gotz, Robert; Dragon, Stefanie

    2003-03-01

    During terminal erythroid differentiation, degradation of RNA is a potential source for nucleotide triphosphates (NTPs) that act as allosteric effectors of hemoglobin. In this investigation, we assessed the developmental profile of RNA and purine/pyrimidine trinucleotides in circulating embryonic chick red blood cells (RBC). Extensive changes of the NTP pattern are observed which differ significantly from what is observed for adult RBC. The biochemical mechanisms have not been identified yet. Therefore, we studied the role of AMP deaminase and IMP/GMP 5'-nucleotidase, which are key enzymes for the regulation of the purine nucleotide pool. Finally, we tested the effect of major NTPs on the oxygen affinity of embryonic/adult hemoglobin. The results are as follows. 1) Together with ATP, UTP and CTP serve as allosteric effectors of hemoglobin. 2) Degradation of erythroid RNA is apparently a major source for NTPs. 3) Developmental changes of nucleotide content depend on the activities of key enzymes (AMP deaminase, IMP/GMP 5'-nucleotidase, and pyrimidine 5'-nucleotidase). 4) Oxygen-dependent hormonal regulation of AMP deaminase adjusts the red cell ATP concentration and therefore the hemoglobin oxygen affinity.

  14. Preliminary Study on the Relationship between cAMP Level and gsp Expression in Cultured Human Pituitary Somatotrophinomas

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to investigate the relationship between abnormal intracellular signal transduction and tumorgenesis of human pituitary somatotrophinomas, the effects of protein kinase A (PKA)-dependent growth hormone (GH) releasing hormone (GHRH) and protein kinase C (PKC)-dependent GH-releasing peptide (GHRP-6) on cAMP production were observed by using cell culture and biochemical methods, and the expression of the gsp oncogene was detected by using PCR and direct sequence assay methods in 11 patients with human pituitary somatotrophinomas. It was found that GHRP-6 exerted significant stimulatory effect on cAMP production by 2 gsp-positive tumors and no effect on the gsp-negative tumors. GHRP-6 could enhance the stimulation of cAMP production induced by GHRH in tumor without gsp oncogenes. It was suggested that both GHRH and GHRP-6 exert identical effects on human pituitary soamtotrophinomas, which was contributed to the cross-talk between the two intracellular signal transduction pathways in pituitary cells.

  15. cAMP-synthesis in a medullary thyroid carcinoma cell line: response to adrenergic agents and prostaglandines.

    Science.gov (United States)

    Mertens, P R; Goretzki, P E; Keck, E

    1999-01-01

    Calcitonin secretion by C-cells is mediated through intracellular 3'5'-cyclic adenosine monophosphate (cAMP) and calcium signaling. Calcitonin release stimulation tests may take advantage of both signaling cascades in screening for medullary thyroid carcinomas (MTC). To elucidate the regulation of the adenylyl cyclase system we have determined cAMP levels of a calcitonin-expressing MTC cell line (RG) after exposure to adrenergic agents and prostaglandines. In early passages (20-30) cAMP concentrations were significantly elevated in RG cells after exposure to beta-adrenergic agents and prostaglandines E1 and E2. In advanced passages (60-80) the beta-adrenergic response was no longer detectable and adrenergic receptors were uncoupled from the adenylyl cyclase complex; while the effect of prostaglandines E1 and E2 remained unaffected. Preincubation with dexamethasone, in a process requiring protein new synthesis, re-established the adrenergic response in later passages, indicating that RG cells dedifferentiated in culture over time. Our in vitro findings suggest that MTC cell dedifferentiation may be accompanied by adrenergic receptor-uncoupling from the adenylate cyclase system and that this process may be reversed by dexamethasone incubation.

  16. Ectopic expression of Dahlia merckii defensin DmAMP1 improves papaya resistance to Phytophthora palmivora by reducing pathogen vigor.

    Science.gov (United States)

    Zhu, Yun J; Agbayani, Ricelle; Moore, Paul H

    2007-06-01

    Phytophthora spp., some of the more important casual agents of plant diseases, are responsible for heavy economic losses worldwide. Plant defensins have been introduced as transgenes into a range of species to increase host resistance to pathogens to which they were originally susceptible. However, the effectiveness and mechanism of interaction of the defensins with Phytophthora spp. have not been clearly characterized in planta. In this study, we expressed the Dahlia merckii defensin, DmAMP1, in papaya (Carica papaya L.), a plant highly susceptible to a root, stem, and fruit rot disease caused by Phytophthora palmivora. Extracts of total leaf proteins from transformed plants inhibited growth of Phytophthora in vitro and discs cut from the leaves of transformed plants inhibited growth of Phytophthora in a bioassay. Results from our greenhouse inoculation experiments demonstrate that expressing the DmAMP1 gene in papaya plants increased resistance against P. palmivora and that this increased resistance was associated with reduced hyphae growth of P. palmivora at the infection sites. The inhibitory effects of DmAMP1 expression in papaya suggest this approach has good potential to impart transgenic resistance against Phytophthora in papaya. PMID:17216480

  17. Cytochemical demonstration of cyclic 3', 5' AMP phosphodiesterase in different tissue of migratory locust (Locusta migratoria migratorioides R.F.).

    Science.gov (United States)

    Benedeczky, I; Rózsa, K S

    1981-01-01

    The localization of cyclic 3', 5'-AMP phosphodiesterase was studied by ultrastructural cytochemical methods in the various tissues of Locusta migratoria. Large amounts of electron dense, granular reaction products were detectable on the surface of the corpora allata and the corpus cardiacum, bound to the basal lamina. In the protocerebral neuropile rather large amounts of reaction products were observed in the processes of the glial cells. A significant number of lead phosphate deposit was found to occur on the membrane of certain large axons, the microtubules of the axons, furthermore on the membrane of several terminals. Reaction product was also observable in certain terminals, bound to the synaptic vesicles and the mitochondria. At the same time, electron dense deposits were not detectable at all on the surface of cerebral neurons. In the case of myocardium, reaction product was only found on the basal lamina and the extracellular surface of the sarcolemma. On the basis of our results it can be stated that the cyclic 3', 5'-AMP phosphodiesterase is detectable by cytochemical methods in different tissues of Locusta migratoria and presumably it fulfils the task of the extracellular cAMP level regulation. PMID:6260713

  18. Sesamin induces melanogenesis by microphthalmia-associated transcription factor and tyrosinase up-regulation via cAMP signaling pathway.

    Science.gov (United States)

    Jiang, Zequn; Li, Shasha; Liu, Yunyi; Deng, Pengyi; Huang, Jianguo; He, Guangyuan

    2011-10-01

    In this study, we confirmed that sesamin, an active lignan isolated from sesame seed and oil, is a novel skin-tanning compound. The melanin content and tyrosinase activity were increased by sesamin in a dose-dependent manner in B16 melanoma cells. The mRNA and protein levels of tyrosinase were also enhanced after the treatment with sesamin. Western blot analysis revealed that sesamin induced and sustained up-regulation of microphthalmia-associated transcription factor (MITF). Sesamin could activate cAMP response element (CRE) binding protein (CREB), but it had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or Akt. Moreover, sesamin activated protein kinase A (PKA) via a cAMP-dependent pathway. Consistent with these results, sesamin-mediated increase of melanin synthesis was reduced significantly by H-89, a PKA inhibitor, but not by SB203580, a p38 MAPK inhibitor or by LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Sesamin-mediated phosphorylation of CREB and induction of MITF and tyrosinase expression were also inhibited by H-89. These findings indicated that sesamin could stimulate melanogenesis in B16 cells via the up-regulation of MITF and tyrosinase, which was, in turn, due to the activation of cAMP signaling. PMID:21896570

  19. Roles of intracellular Ca2+ and cyclic AMP in mast cell histamine release induced by radiographic contrast media.

    Science.gov (United States)

    Saito, Mami; Itoh, Yoshinori; Yano, Takahisa; Sendo, Toshiaki; Goromaru, Takeshi; Sakai, Naoko; Oishi, Ryozo

    2003-04-01

    Mast cell histamine release is considered to be associated with the etiology of anaphylactoid reactions to iodinated radiographic contrast media (RCM). In the present study, the effects of various ionic and non-ionic RCM on histamine release from mast cells were compared, and the possible mechanisms of the histamine release were subsequently determined. Both ionic (ioxaglate and amidotrizoate) and non-ionic (iohexol, ioversol, iomeprol, iopamidol and iotrolan) RCM increased histamine release from the dissociated rat pulmonary cells, whereby ionic materials were more potent than non-ionic agents. There was no significant correlation between the extent of histamine release and the osmolarity of each RCM solution. In addition, hyperosmotic mannitol solution (1000 mOsm/kg) caused no marked histamine release. Thus, it is unlikely that the hyperosmolarity of RCM solutions contributes to the histamine release. RCM also stimulated, but to a lesser extent, the histamine release from rat peritoneal cells. The RCM-induced histamine release from both types of cells was inhibited by dibutyl cyclic AMP or combined treatment with forskolin and 3-isobutyl-1-methylxanthine. Corresponding to these results, RCM markedly reduced the cellular cyclic AMP content. On the other hand, the removal of intracellular but not the extracellular Ca2+ attenuated the RCM-induced mast cell histamine release. From these findings, it is suggested that the decrease in cellular cyclic AMP content and an increase in intracellular Ca2+ contribute at least in part to the RCM-induced mast cell histamine release. PMID:12690428

  20. LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.

    Directory of Open Access Journals (Sweden)

    Maikel Jongsma

    Full Text Available Lysophosphatidic acid (LPA, a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6, showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1 being 10-fold more potent than acyl-LPA(18:1. The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2, LPA(5 and LPA(6 receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5 receptor (GPR92, which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5 as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.