Science.gov

Sample records for advanced multi-physics amp

  1. Integration of Advanced Probabilistic Analysis Techniques with Multi-Physics Models

    SciTech Connect

    Cetiner, Mustafa Sacit; none,; Flanagan, George F.; Poore III, Willis P.; Muhlheim, Michael David

    2014-07-30

    An integrated simulation platform that couples probabilistic analysis-based tools with model-based simulation tools can provide valuable insights for reactive and proactive responses to plant operating conditions. The objective of this work is to demonstrate the benefits of a partial implementation of the Small Modular Reactor (SMR) Probabilistic Risk Assessment (PRA) Detailed Framework Specification through the coupling of advanced PRA capabilities and accurate multi-physics plant models. Coupling a probabilistic model with a multi-physics model will aid in design, operations, and safety by providing a more accurate understanding of plant behavior. This represents the first attempt at actually integrating these two types of analyses for a control system used for operations, on a faster than real-time basis. This report documents the development of the basic communication capability to exchange data with the probabilistic model using Reliability Workbench (RWB) and the multi-physics model using Dymola. The communication pathways from injecting a fault (i.e., failing a component) to the probabilistic and multi-physics models were successfully completed. This first version was tested with prototypic models represented in both RWB and Modelica. First, a simple event tree/fault tree (ET/FT) model was created to develop the software code to implement the communication capabilities between the dynamic-link library (dll) and RWB. A program, written in C#, successfully communicates faults to the probabilistic model through the dll. A systems model of the Advanced Liquid-Metal Reactor–Power Reactor Inherently Safe Module (ALMR-PRISM) design developed under another DOE project was upgraded using Dymola to include proper interfaces to allow data exchange with the control application (ConApp). A program, written in C+, successfully communicates faults to the multi-physics model. The results of the example simulation were successfully plotted.

  2. Advanced Mesh-Enabled Monte carlo capability for Multi-Physics Reactor Analysis

    SciTech Connect

    Wilson, Paul; Evans, Thomas; Tautges, Tim

    2012-12-24

    This project will accumulate high-precision fluxes throughout reactor geometry on a non- orthogonal grid of cells to support multi-physics coupling, in order to more accurately calculate parameters such as reactivity coefficients and to generate multi-group cross sections. This work will be based upon recent developments to incorporate advanced geometry and mesh capability in a modular Monte Carlo toolkit with computational science technology that is in use in related reactor simulation software development. Coupling this capability with production-scale Monte Carlo radiation transport codes can provide advanced and extensible test-beds for these developments. Continuous energy Monte Carlo methods are generally considered to be the most accurate computational tool for simulating radiation transport in complex geometries, particularly neutron transport in reactors. Nevertheless, there are several limitations for their use in reactor analysis. Most significantly, there is a trade-off between the fidelity of results in phase space, statistical accuracy, and the amount of computer time required for simulation. Consequently, to achieve an acceptable level of statistical convergence in high-fidelity results required for modern coupled multi-physics analysis, the required computer time makes Monte Carlo methods prohibitive for design iterations and detailed whole-core analysis. More subtly, the statistical uncertainty is typically not uniform throughout the domain, and the simulation quality is limited by the regions with the largest statistical uncertainty. In addition, the formulation of neutron scattering laws in continuous energy Monte Carlo methods makes it difficult to calculate adjoint neutron fluxes required to properly determine important reactivity parameters. Finally, most Monte Carlo codes available for reactor analysis have relied on orthogonal hexahedral grids for tallies that do not conform to the geometric boundaries and are thus generally not well

  3. Specification of the Advanced Burner Test Reactor Multi-Physics Coupling Demonstration Problem

    SciTech Connect

    Shemon, E. R.; Grudzinski, J. J.; Lee, C. H.; Thomas, J. W.; Yu, Y. Q.

    2015-12-21

    This document specifies the multi-physics nuclear reactor demonstration problem using the SHARP software package developed by NEAMS. The SHARP toolset simulates the key coupled physics phenomena inside a nuclear reactor. The PROTEUS neutronics code models the neutron transport within the system, the Nek5000 computational fluid dynamics code models the fluid flow and heat transfer, and the DIABLO structural mechanics code models structural and mechanical deformation. The three codes are coupled to the MOAB mesh framework which allows feedback from neutronics, fluid mechanics, and mechanical deformation in a compatible format.

  4. Advanced computations of multi-physics, multi-scale effects in beam dynamics

    SciTech Connect

    Amundson, J.F.; Macridin, A.; Spentzouris, P.; Stern, E.G.; /Fermilab

    2009-01-01

    Current state-of-the-art beam dynamics simulations include multiple physical effects and multiple physical length and/or time scales. We present recent developments in Synergia2, an accelerator modeling framework designed for multi-physics, multi-scale simulations. We summarize recent several recent results in multi-physics beam dynamics, including simulations of three Fermilab accelerators: the Tevatron, the Main Injector and the Debuncher. Early accelerator simulations focused on single-particle dynamics. To a first approximation, the forces on the particles in an accelerator beam are dominated by the external fields due to magnets, RF cavities, etc., so the single-particle dynamics are the leading physical effects. Detailed simulations of accelerators must include collective effects such as the space-charge repulsion of the beam particles, the effects of wake fields in the beam pipe walls and beam-beam interactions in colliders. These simulations require the sort of massively parallel computers that have only become available in recent times. We give an overview of the accelerator framework Synergia2, which was designed to take advantage of the capabilities of modern computational resources and enable simulations of multiple physical effects. We also summarize some recent results utilizing Synergia2 and BeamBeam3d, a tool specialized for beam-beam simulations.

  5. Fiscal Year 2011 Infrastructure Refactorizations in AMP

    SciTech Connect

    Berrill, Mark A.; Philip, Bobby; Sampath, Rahul S.; Allu, Srikanth; Barai, Pallab; Cochran, Bill; Clarno, Kevin T.; Dilts, Gary A.

    2011-09-01

    In Fiscal Year 2011 (FY11), the AMP (Advanced MultiPhysics) Nuclear Fuel Performance code [1] went through a thorough review and refactorization based on the lessons-learned from the previous year, in which the version 0.9 of the software was released as a prototype. This report describes the refactorization work that has occurred or is in progress during FY11.

  6. Analysis of Advanced Modular Power Systems (AMPS) for Deep Space Exploration

    NASA Technical Reports Server (NTRS)

    Oeftering, Richard; Soeder, James F.; Beach, Ray

    2014-01-01

    The Advanced Modular Power Systems (AMPS) project is developing a modular approach to spacecraft power systems for exploration beyond Earth orbit. AMPS is intended to meet the need of reducing the cost of design development, test and integration and also reducing the operational logistics cost of supporting exploration missions. AMPS seeks to establish modular power building blocks with standardized electrical, mechanical, thermal and data interfaces that can be applied across multiple exploration vehicles. The presentation discusses the results of a cost analysis that compares the cost of the modular approach against a traditional non-modular approach.

  7. Scalable Methods for Uncertainty Quantification, Data Assimilation and Target Accuracy Assessment for Multi-Physics Advanced Simulation of Light Water Reactors

    NASA Astrophysics Data System (ADS)

    Khuwaileh, Bassam

    High fidelity simulation of nuclear reactors entails large scale applications characterized with high dimensionality and tremendous complexity where various physics models are integrated in the form of coupled models (e.g. neutronic with thermal-hydraulic feedback). Each of the coupled modules represents a high fidelity formulation of the first principles governing the physics of interest. Therefore, new developments in high fidelity multi-physics simulation and the corresponding sensitivity/uncertainty quantification analysis are paramount to the development and competitiveness of reactors achieved through enhanced understanding of the design and safety margins. Accordingly, this dissertation introduces efficient and scalable algorithms for performing efficient Uncertainty Quantification (UQ), Data Assimilation (DA) and Target Accuracy Assessment (TAA) for large scale, multi-physics reactor design and safety problems. This dissertation builds upon previous efforts for adaptive core simulation and reduced order modeling algorithms and extends these efforts towards coupled multi-physics models with feedback. The core idea is to recast the reactor physics analysis in terms of reduced order models. This can be achieved via identifying the important/influential degrees of freedom (DoF) via the subspace analysis, such that the required analysis can be recast by considering the important DoF only. In this dissertation, efficient algorithms for lower dimensional subspace construction have been developed for single physics and multi-physics applications with feedback. Then the reduced subspace is used to solve realistic, large scale forward (UQ) and inverse problems (DA and TAA). Once the elite set of DoF is determined, the uncertainty/sensitivity/target accuracy assessment and data assimilation analysis can be performed accurately and efficiently for large scale, high dimensional multi-physics nuclear engineering applications. Hence, in this work a Karhunen-Loeve (KL

  8. Science based integrated approach to advanced nuclear fuel development - integrated multi-scale multi-physics hierarchical modeling and simulation framework Part III: cladding

    SciTech Connect

    Tome, Carlos N; Caro, J A; Lebensohn, R A; Unal, Cetin; Arsenlis, A; Marian, J; Pasamehmetoglu, K

    2010-01-01

    Advancing the performance of Light Water Reactors, Advanced Nuclear Fuel Cycles, and Advanced Reactors, such as the Next Generation Nuclear Power Plants, requires enhancing our fundamental understanding of fuel and materials behavior under irradiation. The capability to accurately model the nuclear fuel systems to develop predictive tools is critical. Not only are fabrication and performance models needed to understand specific aspects of the nuclear fuel, fully coupled fuel simulation codes are required to achieve licensing of specific nuclear fuel designs for operation. The backbone of these codes, models, and simulations is a fundamental understanding and predictive capability for simulating the phase and microstructural behavior of the nuclear fuel system materials and matrices. In this paper we review the current status of the advanced modeling and simulation of nuclear reactor cladding, with emphasis on what is available and what is to be developed in each scale of the project, how we propose to pass information from one scale to the next, and what experimental information is required for benchmarking and advancing the modeling at each scale level.

  9. Recent Advances in the Discovery of Small Molecules Targeting Exchange Proteins Directly Activated by cAMP (EPAC)

    PubMed Central

    Chen, Haijun; Wild, Christopher; Zhou, Xiaobin; Ye, Na; Cheng, Xiaodong; Zhou, Jia

    2014-01-01

    cAMP is a pivotal second messenger that regulates numerous biological processes under physiological and pathological conditions, including cancer, diabetes, heart failure, inflammation and neurological disorders. In the past, all effects of cAMP were initially believed to be mediated by PKA and cyclic nucleotide-regulated ion channels. Since the discovery of EPAC proteins in 1998, accumulating evidence has demonstrated that the net cellular effects of cAMP are also regulated by EPAC. The pursuit of the biological functions of EPAC has benefited from the development and applications of a growing number of pharmacological probes targeting EPAC proteins. In this Perspective, we seek to provide a concise update on recent advances in the development of chemical entities including various membrane-permeable analogues of cAMP and newly discovered EPAC-specific ligands from high throughput assays and hit-to-lead optimizations. PMID:24256330

  10. AMPED Program Overview

    ScienceCinema

    Gur, Ilan

    2016-07-12

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  11. AMPED Program Overview

    SciTech Connect

    Gur, Ilan

    2014-03-04

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  12. Advanced Mitigation Process (AMP) for Improving Laser Damage Threshold of Fused Silica Optics

    NASA Astrophysics Data System (ADS)

    Ye, Xin; Huang, Jin; Liu, Hongjie; Geng, Feng; Sun, Laixi; Jiang, Xiaodong; Wu, Weidong; Qiao, Liang; Zu, Xiaotao; Zheng, Wanguo

    2016-08-01

    The laser damage precursors in subsurface of fused silica (e.g. photosensitive impurities, scratches and redeposited silica compounds) were mitigated by mineral acid leaching and HF etching with multi-frequency ultrasonic agitation, respectively. The comparison of scratches morphology after static etching and high-frequency ultrasonic agitation etching was devoted in our case. And comparison of laser induce damage resistance of scratched and non-scratched fused silica surfaces after HF etching with high-frequency ultrasonic agitation were also investigated in this study. The global laser induce damage resistance was increased significantly after the laser damage precursors were mitigated in this case. The redeposition of reaction produce was avoided by involving multi-frequency ultrasonic and chemical leaching process. These methods made the increase of laser damage threshold more stable. In addition, there is no scratch related damage initiations found on the samples which were treated by Advanced Mitigation Process.

  13. Advanced Mitigation Process (AMP) for Improving Laser Damage Threshold of Fused Silica Optics

    PubMed Central

    Ye, Xin; Huang, Jin; Liu, Hongjie; Geng, Feng; Sun, Laixi; Jiang, Xiaodong; Wu, Weidong; Qiao, Liang; Zu, Xiaotao; Zheng, Wanguo

    2016-01-01

    The laser damage precursors in subsurface of fused silica (e.g. photosensitive impurities, scratches and redeposited silica compounds) were mitigated by mineral acid leaching and HF etching with multi-frequency ultrasonic agitation, respectively. The comparison of scratches morphology after static etching and high-frequency ultrasonic agitation etching was devoted in our case. And comparison of laser induce damage resistance of scratched and non-scratched fused silica surfaces after HF etching with high-frequency ultrasonic agitation were also investigated in this study. The global laser induce damage resistance was increased significantly after the laser damage precursors were mitigated in this case. The redeposition of reaction produce was avoided by involving multi-frequency ultrasonic and chemical leaching process. These methods made the increase of laser damage threshold more stable. In addition, there is no scratch related damage initiations found on the samples which were treated by Advanced Mitigation Process. PMID:27484188

  14. Advances in High-Fidelity Multi-Physics Simulation Techniques

    DTIC Science & Technology

    2008-01-01

    fluid dynamics with other disciplines also yield a large and typically stiff equation set whose numerical solution mandates the development and...and Electromagnetics . . . . . 3 2.1 Governing Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2 Numerical Technique...discrete equivalent of the governing equations . Thus, the values of the solution vector are localized in a pointwise sense at each node of the mesh. This

  15. Optimization and parallelization of the thermal–hydraulic subchannel code CTF for high-fidelity multi-physics applications

    DOE PAGES

    Salko, Robert K.; Schmidt, Rodney C.; Avramova, Maria N.

    2014-11-23

    This study describes major improvements to the computational infrastructure of the CTF subchannel code so that full-core, pincell-resolved (i.e., one computational subchannel per real bundle flow channel) simulations can now be performed in much shorter run-times, either in stand-alone mode or as part of coupled-code multi-physics calculations. These improvements support the goals of the Department Of Energy Consortium for Advanced Simulation of Light Water Reactors (CASL) Energy Innovation Hub to develop high fidelity multi-physics simulation tools for nuclear energy design and analysis.

  16. Multi-Physics Demonstration Problem with the SHARP Reactor Simulation Toolkit

    SciTech Connect

    Merzari, E.; Shemon, E. R.; Yu, Y. Q.; Thomas, J. W.; Obabko, A.; Jain, Rajeev; Mahadevan, Vijay; Tautges, Timothy; Solberg, Jerome; Ferencz, Robert Mark; Whitesides, R.

    2015-12-21

    This report describes to employ SHARP to perform a first-of-a-kind analysis of the core radial expansion phenomenon in an SFR. This effort required significant advances in the framework Multi-Physics Demonstration Problem with the SHARP Reactor Simulation Toolkit used to drive the coupled simulations, manipulate the mesh in response to the deformation of the geometry, and generate the necessary modified mesh files. Furthermore, the model geometry is fairly complex, and consistent mesh generation for the three physics modules required significant effort. Fully-integrated simulations of a 7-assembly mini-core test problem have been performed, and the results are presented here. Physics models of a full-core model of the Advanced Burner Test Reactor have also been developed for each of the three physics modules. Standalone results of each of the three physics modules for the ABTR are presented here, which provides a demonstration of the feasibility of the fully-integrated simulation.

  17. Multi-Physics Analysis of the Fermilab Booster RF Cavity

    SciTech Connect

    Awida, M.; Reid, J.; Yakovlev, V.; Lebedev, V.; Khabiboulline, T.; Champion, M.; /Fermilab

    2012-05-14

    After about 40 years of operation the RF accelerating cavities in Fermilab Booster need an upgrade to improve their reliability and to increase the repetition rate in order to support a future experimental program. An increase in the repetition rate from 7 to 15 Hz entails increasing the power dissipation in the RF cavities, their ferrite loaded tuners, and HOM dampers. The increased duty factor requires careful modelling for the RF heating effects in the cavity. A multi-physic analysis investigating both the RF and thermal properties of Booster cavity under various operating conditions is presented in this paper.

  18. IMPETUS - Interactive MultiPhysics Environment for Unified Simulations.

    PubMed

    Ha, Vi Q; Lykotrafitis, George

    2016-12-08

    We introduce IMPETUS - Interactive MultiPhysics Environment for Unified Simulations, an object oriented, easy-to-use, high performance, C++ program for three-dimensional simulations of complex physical systems that can benefit a large variety of research areas, especially in cell mechanics. The program implements cross-communication between locally interacting particles and continuum models residing in the same physical space while a network facilitates long-range particle interactions. Message Passing Interface is used for inter-processor communication for all simulations.

  19. Interrogating cyclic AMP signaling using optical approaches.

    PubMed

    Jiang, Jason Y; Falcone, Jeffrey L; Curci, Silvana; Hofer, Aldebaran M

    2017-03-01

    Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.

  20. Cyclic AMP in prokaryotes.

    PubMed Central

    Botsford, J L; Harman, J G

    1992-01-01

    Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth. PMID:1315922

  1. Solid Oxide Fuel Cell - Multi-Physics and GUI

    SciTech Connect

    2013-10-10

    SOFC-MP is a simulation tool developed at PNNL to evaluate the tightly coupled multi-physical phenomena in SOFCs. The purpose of the tool is to allow SOFC manufacturers to numerically test changes in planar stack design to meet DOE technical targets. The SOFC-MP 2D module is designed for computational efficiency to enable rapid engineering evaluations for operation of tall symmetric stacks. It can quickly compute distributions for the current density, voltage, temperature, and species composition in tall stacks with co-flow or counter-flow orientations. The 3D module computes distributions in entire 3D domain and handles all planner configurations: co-flow, counter-flow, and cross-flow. The detailed data from 3D simulation can be used as input for structural analysis. SOFC-MP GUI integrates both 2D and 3D modules, and it provides user friendly pre-processing and post-processing capabilities.

  2. A Global Sensitivity Analysis Methodology for Multi-physics Applications

    SciTech Connect

    Tong, C H; Graziani, F R

    2007-02-02

    Experiments are conducted to draw inferences about an entire ensemble based on a selected number of observations. This applies to both physical experiments as well as computer experiments, the latter of which are performed by running the simulation models at different input configurations and analyzing the output responses. Computer experiments are instrumental in enabling model analyses such as uncertainty quantification and sensitivity analysis. This report focuses on a global sensitivity analysis methodology that relies on a divide-and-conquer strategy and uses intelligent computer experiments. The objective is to assess qualitatively and/or quantitatively how the variabilities of simulation output responses can be accounted for by input variabilities. We address global sensitivity analysis in three aspects: methodology, sampling/analysis strategies, and an implementation framework. The methodology consists of three major steps: (1) construct credible input ranges; (2) perform a parameter screening study; and (3) perform a quantitative sensitivity analysis on a reduced set of parameters. Once identified, research effort should be directed to the most sensitive parameters to reduce their uncertainty bounds. This process is repeated with tightened uncertainty bounds for the sensitive parameters until the output uncertainties become acceptable. To accommodate the needs of multi-physics application, this methodology should be recursively applied to individual physics modules. The methodology is also distinguished by an efficient technique for computing parameter interactions. Details for each step will be given using simple examples. Numerical results on large scale multi-physics applications will be available in another report. Computational techniques targeted for this methodology have been implemented in a software package called PSUADE.

  3. A theory manual for multi-physics code coupling in LIME.

    SciTech Connect

    Belcourt, Noel; Bartlett, Roscoe Ainsworth; Pawlowski, Roger Patrick; Schmidt, Rodney Cannon; Hooper, Russell Warren

    2011-03-01

    The Lightweight Integrating Multi-physics Environment (LIME) is a software package for creating multi-physics simulation codes. Its primary application space is when computer codes are currently available to solve different parts of a multi-physics problem and now need to be coupled with other such codes. In this report we define a common domain language for discussing multi-physics coupling and describe the basic theory associated with multiphysics coupling algorithms that are to be supported in LIME. We provide an assessment of coupling techniques for both steady-state and time dependent coupled systems. Example couplings are also demonstrated.

  4. Lithium-Ion Battery Safety Study Using Multi-Physics Internal Short-Circuit Model (Presentation)

    SciTech Connect

    Kim, G-.H.; Smith, K.; Pesaran, A.

    2009-06-01

    This presentation outlines NREL's multi-physics simulation study to characterize an internal short by linking and integrating electrochemical cell, electro-thermal, and abuse reaction kinetics models.

  5. Modelling transport phenomena in a multi-physics context

    NASA Astrophysics Data System (ADS)

    Marra, Francesco

    2015-01-01

    Innovative heating research on cooking, pasteurization/sterilization, defrosting, thawing and drying, often focuses on areas which include the assessment of processing time, evaluation of heating uniformity, studying the impact on quality attributes of the final product as well as considering the energy efficiency of these heating processes. During the last twenty years, so-called electro-heating-processes (radio-frequency - RF, microwaves - MW and ohmic - OH) gained a wide interest in industrial food processing and many applications using the above mentioned technologies have been developed with the aim of reducing processing time, improving process efficiency and, in many cases, the heating uniformity. In the area of innovative heating, electro-heating accounts for a considerable portion of both the scientific literature and commercial applications, which can be subdivided into either direct electro-heating (as in the case of OH heating) where electrical current is applied directly to the food or indirect electro-heating (e.g. MW and RF heating) where the electrical energy is firstly converted to electromagnetic radiation which subsequently generates heat within a product. New software packages, which make easier solution of PDEs based mathematical models, and new computers, capable of larger RAM and more efficient CPU performances, allowed an increasing interest about modelling transport phenomena in systems and processes - as the ones encountered in food processing - that can be complex in terms of geometry, composition, boundary conditions but also - as in the case of electro-heating assisted applications - in terms of interaction with other physical phenomena such as displacement of electric or magnetic field. This paper deals with the description of approaches used in modelling transport phenomena in a multi-physics context such as RF, MW and OH assisted heating.

  6. Modelling transport phenomena in a multi-physics context

    SciTech Connect

    Marra, Francesco

    2015-01-22

    Innovative heating research on cooking, pasteurization/sterilization, defrosting, thawing and drying, often focuses on areas which include the assessment of processing time, evaluation of heating uniformity, studying the impact on quality attributes of the final product as well as considering the energy efficiency of these heating processes. During the last twenty years, so-called electro-heating-processes (radio-frequency - RF, microwaves - MW and ohmic - OH) gained a wide interest in industrial food processing and many applications using the above mentioned technologies have been developed with the aim of reducing processing time, improving process efficiency and, in many cases, the heating uniformity. In the area of innovative heating, electro-heating accounts for a considerable portion of both the scientific literature and commercial applications, which can be subdivided into either direct electro-heating (as in the case of OH heating) where electrical current is applied directly to the food or indirect electro-heating (e.g. MW and RF heating) where the electrical energy is firstly converted to electromagnetic radiation which subsequently generates heat within a product. New software packages, which make easier solution of PDEs based mathematical models, and new computers, capable of larger RAM and more efficient CPU performances, allowed an increasing interest about modelling transport phenomena in systems and processes - as the ones encountered in food processing - that can be complex in terms of geometry, composition, boundary conditions but also - as in the case of electro-heating assisted applications - in terms of interaction with other physical phenomena such as displacement of electric or magnetic field. This paper deals with the description of approaches used in modelling transport phenomena in a multi-physics context such as RF, MW and OH assisted heating.

  7. Agile manufacturing prototyping system (AMPS)

    SciTech Connect

    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.

  8. Metformin inhibits advanced glycation end products (AGEs)-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing AGEs receptor expression via AMP-activated protein kinase.

    PubMed

    Ishibashi, Y; Matsui, T; Takeuchi, M; Yamagishi, S

    2013-05-01

    Metformin use has been reported to decrease breast cancer incidence and mortality in diabetic patients. We have previously shown that advanced glycation end products (AGEs) and their receptor (RAGE) interaction stimulate growth and/or migration of pancreatic cancer and melanoma cells. However, effects of metformin on AGEs-RAGE axis in breast cancers remain unknown. We examined here whether and how metformin could block the AGEs-induced growth and vascular endothelial growth factor (VEGF) expression in MCF-7 breast cancer cells. Cell proliferation was measured with an electron coupling reagent WST-1 based colorimetric assay. Gene expression level was evaluated by real-time reverse-transcription polymerase chain reactions. AGEs significantly increased cell proliferation of MCF-7 cells, which was completely prevented by the treatment with 0.01 or 0.1 mM metformin or anti-RAGE antibodies. Furthermore, metformin at 0.01 mM completely suppressed the AGEs-induced upregulation of RAGE and VEGF mRNA levels in MCF-7 cells. An inhibitor of AMP-activated protein kinase, compound C significantly blocked the growth-inhibitory and RAGE and VEGF suppressing effects of metformin in AGEs-exposed MCF-7 cells. Our present study suggests that metformin could inhibit the AGEs-induced growth and VEGF expression in MCF-7 breast cancer cells by suppressing RAGE gene expression via AMP-activated protein kinase pathway. Metformin may protect against breast cancer expansion in diabetic patients by blocking the AGEs-RAGE axis.

  9. cAMP and Mitochondria

    PubMed Central

    Valsecchi, Federica; Ramos-Espiritu, Lavoisier S.; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Phosphorylation of mitochondrial proteins has emerged as a major regulatory mechanism for metabolic adaptation. cAMP signaling and PKA phosphorylation of mitochondrial proteins have just started to be investigated, and the presence of cAMP-generating enzymes and PKA inside mitochondria is still controversial. Here, we discuss the role of cAMP in regulating mitochondrial bioenergetics through protein phosphorylation and the evidence for soluble adenylyl cyclase as the source of cAMP inside mitochondria. PMID:23636265

  10. A lightweight in situ visualization and analysis infrastructure for multi-physics HPC simulation codes

    SciTech Connect

    Harrison, Cyrus; Larsen, Matt; Brugger, Eric

    2016-12-05

    Strawman is a system designed to explore the in situ visualization and analysis needs of simulation code teams running multi-physics calculations on many-core HPC architectures. It porvides rendering pipelines that can leverage both many-core CPUs and GPUs to render images of simulation meshes.

  11. A multi-physical model of actuation response in dielectric gels

    NASA Astrophysics Data System (ADS)

    Li, Bo; Chang, LongFei; Asaka, Kinji; Chen, Hualing; Li, Dichen

    2016-12-01

    Actuation deformation of a dielectric gel is attributed to: the solvent diffusion, the electrical polarization and material hyperelasticity. A multi-physical model, coupling electrical and mechanical quantities, is established, based on the thermodynamics. A set of constitutive relations is derived as an equation of state for characterization. The model is applied to specific cases as effective validations. Physical and chemical parameters affect the performance of the gel, showing nonlinear deformation and instability. This model offers guidance for engineering application.

  12. Multi-physics design and analyses of long life reactors for lunar outposts

    NASA Astrophysics Data System (ADS)

    Schriener, Timothy M.

    event of a launch abort accident. Increasing the amount of fuel in the reactor core, and hence its operational life, would be possible by launching the reactor unfueled and fueling it on the Moon. Such a reactor would, thus, not be subject to launch criticality safety requirements. However, loading the reactor with fuel on the Moon presents a challenge, requiring special designs of the core and the fuel elements, which lend themselves to fueling on the lunar surface. This research investigates examples of both a solid core reactor that would be fueled at launch as well as an advanced concept which could be fueled on the Moon. Increasing the operational life of a reactor fueled at launch is exercised for the NaK-78 cooled Sectored Compact Reactor (SCoRe). A multi-physics design and analyses methodology is developed which iteratively couples together detailed Monte Carlo neutronics simulations with 3-D Computational Fluid Dynamics (CFD) and thermal-hydraulics analyses. Using this methodology the operational life of this compact, fast spectrum reactor is increased by reconfiguring the core geometry to reduce neutron leakage and parasitic absorption, for the same amount of HEU in the core, and meeting launch safety requirements. The multi-physics analyses determine the impacts of the various design changes on the reactor's neutronics and thermal-hydraulics performance. The option of increasing the operational life of a reactor by loading it on the Moon is exercised for the Pellet Bed Reactor (PeBR). The PeBR uses spherical fuel pellets and is cooled by He-Xe gas, allowing the reactor core to be loaded with fuel pellets and charged with working fluid on the lunar surface. The performed neutronics analyses ensure the PeBR design achieves a long operational life, and develops safe launch canister designs to transport the spherical fuel pellets to the lunar surface. The research also investigates loading the PeBR core with fuel pellets on the Moon using a transient Discrete

  13. Numerical Stability and Accuracy of Temporally Coupled Multi-Physics Modules in Wind-Turbine CAE Tools

    SciTech Connect

    Gasmi, A.; Sprague, M. A.; Jonkman, J. M.; Jones, W. B.

    2013-02-01

    In this paper we examine the stability and accuracy of numerical algorithms for coupling time-dependent multi-physics modules relevant to computer-aided engineering (CAE) of wind turbines. This work is motivated by an in-progress major revision of FAST, the National Renewable Energy Laboratory's (NREL's) premier aero-elastic CAE simulation tool. We employ two simple examples as test systems, while algorithm descriptions are kept general. Coupled-system governing equations are framed in monolithic and partitioned representations as differential-algebraic equations. Explicit and implicit loose partition coupling is examined. In explicit coupling, partitions are advanced in time from known information. In implicit coupling, there is dependence on other-partition data at the next time step; coupling is accomplished through a predictor-corrector (PC) approach. Numerical time integration of coupled ordinary-differential equations (ODEs) is accomplished with one of three, fourth-order fixed-time-increment methods: Runge-Kutta (RK), Adams-Bashforth (AB), and Adams-Bashforth-Moulton (ABM). Through numerical experiments it is shown that explicit coupling can be dramatically less stable and less accurate than simulations performed with the monolithic system. However, PC implicit coupling restored stability and fourth-order accuracy for ABM; only second-order accuracy was achieved with RK integration. For systems without constraints, explicit time integration with AB and explicit loose coupling exhibited desired accuracy and stability.

  14. Experiment definition studies for AMPS Spacelab

    NASA Technical Reports Server (NTRS)

    Liemohn, H.

    1975-01-01

    The electrical charging of the space shuttle orbiter is discussed in relation to the AMPS Spacelab payload along with an operations research technique for the selection of AMPS Spacelab experiments. Experiments proposed for AMPS include: hydromagnetic wave experiments; bistatic sounder of AMPS wake; and an artificial meteor gun. Experiment objectives and instrument functions are given for all experiments.

  15. Module-based Hybrid Uncertainty Quantification for Multi-physics Applications: Theory and Software

    SciTech Connect

    Tong, Charles; Chen, Xiao; Iaccarino, Gianluca; Mittal, Akshay

    2013-10-08

    In this project we proposed to develop an innovative uncertainty quantification methodology that captures the best of the two competing approaches in UQ, namely, intrusive and non-intrusive approaches. The idea is to develop the mathematics and the associated computational framework and algorithms to facilitate the use of intrusive or non-intrusive UQ methods in different modules of a multi-physics multi-module simulation model in a way that physics code developers for different modules are shielded (as much as possible) from the chores of accounting for the uncertain ties introduced by the other modules. As the result of our research and development, we have produced a number of publications, conference presentations, and a software product.

  16. Multi-Physics Markov Chain Monte Carlo Methods for Subsurface Flows

    NASA Astrophysics Data System (ADS)

    Rigelo, J.; Ginting, V.; Rahunanthan, A.; Pereira, F.

    2014-12-01

    For CO2 sequestration in deep saline aquifers, contaminant transport in subsurface, and oil or gas recovery, we often need to forecast flow patterns. Subsurface characterization is a critical and challenging step in flow forecasting. To characterize subsurface properties we establish a statistical description of the subsurface properties that are conditioned to existing dynamic and static data. A Markov Chain Monte Carlo (MCMC) algorithm is used in a Bayesian statistical description to reconstruct the spatial distribution of rock permeability and porosity. The MCMC algorithm requires repeatedly solving a set of nonlinear partial differential equations describing displacement of fluids in porous media for different values of permeability and porosity. The time needed for the generation of a reliable MCMC chain using the algorithm can be too long to be practical for flow forecasting. In this work we develop fast and effective computational methods for generating MCMC chains in the Bayesian framework for the subsurface characterization. Our strategy consists of constructing a family of computationally inexpensive preconditioners based on simpler physics as well as on surrogate models such that the number of fine-grid simulations is drastically reduced in the generated MCMC chains. In particular, we introduce a huff-puff technique as screening step in a three-stage multi-physics MCMC algorithm to reduce the number of expensive final stage simulations. The huff-puff technique in the algorithm enables a better characterization of subsurface near wells. We assess the quality of the proposed multi-physics MCMC methods by considering Monte Carlo simulations for forecasting oil production in an oil reservoir.

  17. Optimization and Parallelization of the Thermal-Hydraulic Sub-channel Code CTF for High-Fidelity Multi-physics Applications

    SciTech Connect

    Salko, Robert K; Schmidt, Rodney; Avramova, Maria N

    2014-01-01

    This paper describes major improvements to the computational infrastructure of the CTF sub-channel code so that full-core sub-channel-resolved simulations can now be performed in much shorter run-times, either in stand-alone mode or as part of coupled-code multi-physics calculations. These improvements support the goals of the Department Of Energy (DOE) Consortium for Advanced Simulations of Light Water (CASL) Energy Innovation Hub to develop high fidelity multi-physics simulation tools for nuclear energy design and analysis. A set of serial code optimizations--including fixing computational inefficiencies, optimizing the numerical approach, and making smarter data storage choices--are first described and shown to reduce both execution time and memory usage by about a factor of ten. Next, a Single Program Multiple Data (SPMD) parallelization strategy targeting distributed memory Multiple Instruction Multiple Data (MIMD) platforms and utilizing domain-decomposition is presented. In this approach, data communication between processors is accomplished by inserting standard MPI calls at strategic points in the code. The domain decomposition approach implemented assigns one MPI process to each fuel assembly, with each domain being represented by its own CTF input file. The creation of CTF input files, both for serial and parallel runs, is also fully automated through use of a pre-processor utility that takes a greatly reduced set of user input over the traditional CTF input file. To run CTF in parallel, two additional libraries are currently needed; MPI, for inter-processor message passing, and the Parallel Extensible Toolkit for Scientific Computation (PETSc), which is leveraged to solve the global pressure matrix in parallel. Results presented include a set of testing and verification calculations and performance tests assessing parallel scaling characteristics up to a full core, sub-channel-resolved model of Watts Bar Unit 1 under hot full-power conditions (193 17x17

  18. The cAMP analogs have potent anti-proliferative effects on medullary thyroid cancer cell lines.

    PubMed

    Dicitore, Alessandra; Grassi, Elisa Stellaria; Caraglia, Michele; Borghi, Maria Orietta; Gaudenzi, Germano; Hofland, Leo J; Persani, Luca; Vitale, Giovanni

    2016-01-01

    The oncogenic activation of the rearranged during transfection (RET) proto-oncogene has a main role in the pathogenesis of medullary thyroid cancer (MTC). Several lines of evidence suggest that RET function could be influenced by cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity. We evaluated the in vitro anti-tumor activity of 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) and PKA type I-selective cAMP analogs [equimolar combination of the 8-piperidinoadenosine-3',5'-cyclic monophosphate (8-PIP-cAMP) and 8-hexylaminoadenosine-3',5'-cyclic monophosphate (8-HA-cAMP) in MTC cell lines (TT and MZ-CRC-1)]. 8-Cl-cAMP and the PKA I-selective cAMP analogs showed a potent anti-proliferative effect in both cell lines. In detail, 8-Cl-cAMP blocked significantly the transition of TT cell population from G2/M to G0/G1 phase and from G0/G1 to S phase and of MZ-CRC-1 cells from G0/G1 to S phase. Moreover, 8-Cl-cAMP induced apoptosis in both cell lines, as demonstrated by FACS analysis for annexin V-FITC/propidium iodide, the activation of caspase-3 and PARP cleavage. On the other hand, the only effect induced by PKA I-selective cAMP analogs was a delay in G0/G1-S and S-G2/M progression in TT and MZ-CRC-1 cells, respectively. In conclusion, these data demonstrate that cAMP analogs, particularly 8-Cl-cAMP, significantly suppress in vitro MTC proliferation and provide rationale for a potential clinical use of cAMP analogs in the treatment of advanced MTC.

  19. Data-driven prognosis: a multi-physics approach verified via balloon burst experiment

    PubMed Central

    Chandra, Abhijit; Kar, Oliva

    2015-01-01

    A multi-physics formulation for data-driven prognosis (DDP) is developed. Unlike traditional predictive strategies that require controlled offline measurements or ‘training’ for determination of constitutive parameters to derive the transitional statistics, the proposed DDP algorithm relies solely on in situ measurements. It uses a deterministic mechanics framework, but the stochastic nature of the solution arises naturally from the underlying assumptions regarding the order of the conservation potential as well as the number of dimensions involved. The proposed DDP scheme is capable of predicting onset of instabilities. Because the need for offline testing (or training) is obviated, it can be easily implemented for systems where such a priori testing is difficult or even impossible to conduct. The prognosis capability is demonstrated here via a balloon burst experiment where the instability is predicted using only online visual observations. The DDP scheme never failed to predict the incipient failure, and no false-positives were issued. The DDP algorithm is applicable to other types of datasets. Time horizons of DDP predictions can be adjusted by using memory over different time windows. Thus, a big dataset can be parsed in time to make a range of predictions over varying time horizons. PMID:27547071

  20. Data-driven prognosis: a multi-physics approach verified via balloon burst experiment.

    PubMed

    Chandra, Abhijit; Kar, Oliva

    2015-04-08

    A multi-physics formulation for data-driven prognosis (DDP) is developed. Unlike traditional predictive strategies that require controlled offline measurements or 'training' for determination of constitutive parameters to derive the transitional statistics, the proposed DDP algorithm relies solely on in situ measurements. It uses a deterministic mechanics framework, but the stochastic nature of the solution arises naturally from the underlying assumptions regarding the order of the conservation potential as well as the number of dimensions involved. The proposed DDP scheme is capable of predicting onset of instabilities. Because the need for offline testing (or training) is obviated, it can be easily implemented for systems where such a priori testing is difficult or even impossible to conduct. The prognosis capability is demonstrated here via a balloon burst experiment where the instability is predicted using only online visual observations. The DDP scheme never failed to predict the incipient failure, and no false-positives were issued. The DDP algorithm is applicable to other types of datasets. Time horizons of DDP predictions can be adjusted by using memory over different time windows. Thus, a big dataset can be parsed in time to make a range of predictions over varying time horizons.

  1. A self-taught artificial agent for multi-physics computational model personalization.

    PubMed

    Neumann, Dominik; Mansi, Tommaso; Itu, Lucian; Georgescu, Bogdan; Kayvanpour, Elham; Sedaghat-Hamedani, Farbod; Amr, Ali; Haas, Jan; Katus, Hugo; Meder, Benjamin; Steidl, Stefan; Hornegger, Joachim; Comaniciu, Dorin

    2016-12-01

    Personalization is the process of fitting a model to patient data, a critical step towards application of multi-physics computational models in clinical practice. Designing robust personalization algorithms is often a tedious, time-consuming, model- and data-specific process. We propose to use artificial intelligence concepts to learn this task, inspired by how human experts manually perform it. The problem is reformulated in terms of reinforcement learning. In an off-line phase, Vito, our self-taught artificial agent, learns a representative decision process model through exploration of the computational model: it learns how the model behaves under change of parameters. The agent then automatically learns an optimal strategy for on-line personalization. The algorithm is model-independent; applying it to a new model requires only adjusting few hyper-parameters of the agent and defining the observations to match. The full knowledge of the model itself is not required. Vito was tested in a synthetic scenario, showing that it could learn how to optimize cost functions generically. Then Vito was applied to the inverse problem of cardiac electrophysiology and the personalization of a whole-body circulation model. The obtained results suggested that Vito could achieve equivalent, if not better goodness of fit than standard methods, while being more robust (up to 11% higher success rates) and with faster (up to seven times) convergence rate. Our artificial intelligence approach could thus make personalization algorithms generalizable and self-adaptable to any patient and any model.

  2. A novel medical image data-based multi-physics simulation platform for computational life sciences.

    PubMed

    Neufeld, Esra; Szczerba, Dominik; Chavannes, Nicolas; Kuster, Niels

    2013-04-06

    Simulating and modelling complex biological systems in computational life sciences requires specialized software tools that can perform medical image data-based modelling, jointly visualize the data and computational results, and handle large, complex, realistic and often noisy anatomical models. The required novel solvers must provide the power to model the physics, biology and physiology of living tissue within the full complexity of the human anatomy (e.g. neuronal activity, perfusion and ultrasound propagation). A multi-physics simulation platform satisfying these requirements has been developed for applications including device development and optimization, safety assessment, basic research, and treatment planning. This simulation platform consists of detailed, parametrized anatomical models, a segmentation and meshing tool, a wide range of solvers and optimizers, a framework for the rapid development of specialized and parallelized finite element method solvers, a visualization toolkit-based visualization engine, a Python scripting interface for customized applications, a coupling framework, and more. Core components are cross-platform compatible and use open formats. Several examples of applications are presented: hyperthermia cancer treatment planning, tumour growth modelling, evaluating the magneto-haemodynamic effect as a biomarker and physics-based morphing of anatomical models.

  3. A multi-physical model for charge and mass transport in a flexible ionic polymer sensor

    NASA Astrophysics Data System (ADS)

    Zhu, Zicai; Asaka, Kinji; Takagi, Kentaro; Aabloo, Alvo; Horiuchi, Tetsuya

    2016-04-01

    An ionic polymer material can generate electrical potential and function as a bio-sensor under a non-uniform deformation. Ionic polymer-metal composite (IPMC) is a typical flexible ionic polymer sensor material. A multi-physical sensing model is presented at first based on the same physical equations in the physical model for IPMC actuator we obtained before. Under an applied bending deformation, water and cation migrate to the direction of outside electrode immediately. Redistribution of cations causes an electrical potential difference between two electrodes. The cation migration is strongly restrained by the generated electrical potential. And the migrated cations will move back to the inner electrode under the concentration diffusion effect and lead to a relaxation of electrical potential. In the whole sensing process, transport and redistribution of charge and mass are revealed along the thickness direction by numerical analysis. The sensing process is a revised physical process of the actuation, however, the transport properties are quite different from those of the later. And the effective dielectric constant of IPMC, which is related to the morphology of the electrode-ionic polymer interface, is proved to have little relation with the sensing amplitude. All the conclusions are significant for ionic polymer sensing material design.

  4. Partitioned coupling strategies for multi-physically coupled radiative heat transfer problems

    SciTech Connect

    Wendt, Gunnar; Erbts, Patrick Düster, Alexander

    2015-11-01

    This article aims to propose new aspects concerning a partitioned solution strategy for multi-physically coupled fields including the physics of thermal radiation. Particularly, we focus on the partitioned treatment of electro–thermo-mechanical problems with an additional fourth thermal radiation field. One of the main goals is to take advantage of the flexibility of the partitioned approach to enable combinations of different simulation software and solvers. Within the frame of this article, we limit ourselves to the case of nonlinear thermoelasticity at finite strains, using temperature-dependent material parameters. For the thermal radiation field, diffuse radiating surfaces and gray participating media are assumed. Moreover, we present a robust and fast partitioned coupling strategy for the fourth field problem. Stability and efficiency of the implicit coupling algorithm are improved drawing on several methods to stabilize and to accelerate the convergence. To conclude and to review the effectiveness and the advantages of the additional thermal radiation field several numerical examples are considered to study the proposed algorithm. In particular we focus on an industrial application, namely the electro–thermo-mechanical modeling of the field-assisted sintering technology.

  5. DAG Software Architectures for Multi-Scale Multi-Physics Problems at Petascale and Beyond

    NASA Astrophysics Data System (ADS)

    Berzins, Martin

    2015-03-01

    The challenge of computations at Petascale and beyond is to ensure how to make possible efficient calculations on possibly hundreds of thousands for cores or on large numbers of GPUs or Intel Xeon Phis. An important methodology for achieving this is at present thought to be that of asynchronous task-based parallelism. The success of this approach will be demonstrated using the Uintah software framework for the solution of coupled fluid-structure interaction problems with chemical reactions. The layered approach of this software makes it possible for the user to specify the physical problems without parallel code, for that specification to be translated into a parallel set of tasks. These tasks are executed using a runtime system that executes tasks asynchronously and sometimes out-of-order. The scalability and portability of this approach will be demonstrated using examples from large scale combustion problems, industrial detonations and multi-scale, multi-physics models. The challenges of scaling such calculations to the next generations of leadership class computers (with more than a hundred petaflops) will be discussed. Thanks to NSF, XSEDE, DOE NNSA, DOE NETL, DOE ALCC and DOE INCITE.

  6. A novel medical image data-based multi-physics simulation platform for computational life sciences

    PubMed Central

    Neufeld, Esra; Szczerba, Dominik; Chavannes, Nicolas; Kuster, Niels

    2013-01-01

    Simulating and modelling complex biological systems in computational life sciences requires specialized software tools that can perform medical image data-based modelling, jointly visualize the data and computational results, and handle large, complex, realistic and often noisy anatomical models. The required novel solvers must provide the power to model the physics, biology and physiology of living tissue within the full complexity of the human anatomy (e.g. neuronal activity, perfusion and ultrasound propagation). A multi-physics simulation platform satisfying these requirements has been developed for applications including device development and optimization, safety assessment, basic research, and treatment planning. This simulation platform consists of detailed, parametrized anatomical models, a segmentation and meshing tool, a wide range of solvers and optimizers, a framework for the rapid development of specialized and parallelized finite element method solvers, a visualization toolkit-based visualization engine, a Python scripting interface for customized applications, a coupling framework, and more. Core components are cross-platform compatible and use open formats. Several examples of applications are presented: hyperthermia cancer treatment planning, tumour growth modelling, evaluating the magneto-haemodynamic effect as a biomarker and physics-based morphing of anatomical models. PMID:24427518

  7. Research on Structural Safety of the Stratospheric Airship Based on Multi-Physics Coupling Calculation

    NASA Astrophysics Data System (ADS)

    Ma, Z.; Hou, Z.; Zang, X.

    2015-09-01

    As a large-scale flexible inflatable structure by a huge inner lifting gas volume of several hundred thousand cubic meters, the stratospheric airship's thermal characteristic of inner gas plays an important role in its structural performance. During the floating flight, the day-night variation of the combined thermal condition leads to the fluctuation of the flow field inside the airship, which will remarkably affect the pressure acted on the skin and the structural safety of the stratospheric airship. According to the multi-physics coupling mechanism mentioned above, a numerical procedure of structural safety analysis of stratospheric airships is developed and the thermal model, CFD model, finite element code and criterion of structural strength are integrated. Based on the computation models, the distributions of the deformations and stresses of the skin are calculated with the variation of day-night time. The effects of loads conditions and structural configurations on the structural safety of stratospheric airships in the floating condition are evaluated. The numerical results can be referenced for the structural design of stratospheric airships.

  8. Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 2: novel specific binding sites in hepatopancreas and cAMP as a second messenger

    PubMed Central

    Zmora, Nilli; Sagi, Amir; Zohar, Yonathan; Chung, J Sook

    2009-01-01

    The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than that of juvenile YO membranes (KD values: 3.22 × 10-8 and 4.19 × 10-10 M/mg protein, respectively). The number of maximum binding sites (BMAX) was approximately two times higher in the hepatopancreas than in the YO (BMAX values: 9.24 × 10-9 and 4.8 × 10-9 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [125I] MIH or [125I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt. PMID:19583849

  9. Amps particle accelerator definition study

    NASA Technical Reports Server (NTRS)

    Sellen, J. M., Jr.

    1975-01-01

    The Particle Accelerator System of the AMPS (Atmospheric, Magnetospheric, and Plasmas in Space) payload is a series of charged particle accelerators to be flown with the Space Transportation System Shuttle on Spacelab missions. In the configuration presented, the total particle accelerator system consists of an energetic electron beam, an energetic ion accelerator, and both low voltage and high voltage plasma acceleration devices. The Orbiter is illustrated with such a particle accelerator system.

  10. Towards a multi-physics modelling framework for thrombolysis under the influence of blood flow.

    PubMed

    Piebalgs, Andris; Xu, X Yun

    2015-12-06

    Thrombolytic therapy is an effective means of treating thromboembolic diseases but can also give rise to life-threatening side effects. The infusion of a high drug concentration can provoke internal bleeding while an insufficient dose can lead to artery reocclusion. It is hoped that mathematical modelling of the process of clot lysis can lead to a better understanding and improvement of thrombolytic therapy. To this end, a multi-physics continuum model has been developed to simulate the dissolution of clot over time upon the addition of tissue plasminogen activator (tPA). The transport of tPA and other lytic proteins is modelled by a set of reaction-diffusion-convection equations, while blood flow is described by volume-averaged continuity and momentum equations. The clot is modelled as a fibrous porous medium with its properties being determined as a function of the fibrin fibre radius and voidage of the clot. A unique feature of the model is that it is capable of simulating the entire lytic process from the initial phase of lysis of an occlusive thrombus (diffusion-limited transport), the process of recanalization, to post-canalization thrombolysis under the influence of convective blood flow. The model has been used to examine the dissolution of a fully occluding clot in a simplified artery at different pressure drops. Our predicted lytic front velocities during the initial stage of lysis agree well with experimental and computational results reported by others. Following canalization, clot lysis patterns are strongly influenced by local flow patterns, which are symmetric at low pressure drops, but asymmetric at higher pressure drops, which give rise to larger recirculation regions and extended areas of intense drug accumulation.

  11. Towards a multi-physics modelling framework for thrombolysis under the influence of blood flow

    PubMed Central

    Piebalgs, Andris

    2015-01-01

    Thrombolytic therapy is an effective means of treating thromboembolic diseases but can also give rise to life-threatening side effects. The infusion of a high drug concentration can provoke internal bleeding while an insufficient dose can lead to artery reocclusion. It is hoped that mathematical modelling of the process of clot lysis can lead to a better understanding and improvement of thrombolytic therapy. To this end, a multi-physics continuum model has been developed to simulate the dissolution of clot over time upon the addition of tissue plasminogen activator (tPA). The transport of tPA and other lytic proteins is modelled by a set of reaction–diffusion–convection equations, while blood flow is described by volume-averaged continuity and momentum equations. The clot is modelled as a fibrous porous medium with its properties being determined as a function of the fibrin fibre radius and voidage of the clot. A unique feature of the model is that it is capable of simulating the entire lytic process from the initial phase of lysis of an occlusive thrombus (diffusion-limited transport), the process of recanalization, to post-canalization thrombolysis under the influence of convective blood flow. The model has been used to examine the dissolution of a fully occluding clot in a simplified artery at different pressure drops. Our predicted lytic front velocities during the initial stage of lysis agree well with experimental and computational results reported by others. Following canalization, clot lysis patterns are strongly influenced by local flow patterns, which are symmetric at low pressure drops, but asymmetric at higher pressure drops, which give rise to larger recirculation regions and extended areas of intense drug accumulation. PMID:26655469

  12. Multi-physics simulation of metal printing at micro/nanoscale using meniscus-confined electrodeposition: Effect of environmental humidity

    NASA Astrophysics Data System (ADS)

    Morsali, Seyedreza; Daryadel, Soheil; Zhou, Zhong; Behroozfar, Ali; Qian, Dong; Minary-Jolandan, Majid

    2017-01-01

    Capability to print metals at micro/nanoscale in arbitrary 3D patterns at local points of interest will have applications in nano-electronics and sensors. Meniscus-confined electrodeposition (MCED) is a manufacturing process that enables depositing metals from an electrolyte containing nozzle (pipette) in arbitrary 3D patterns. In this process, a meniscus (liquid bridge or capillary) between the pipette tip and the substrate governs the localized electrodeposition process. Fabrication of metallic microstructures using this process is a multi-physics process in which electrodeposition, fluid dynamics, and mass and heat transfer physics are simultaneously involved. We utilized multi-physics finite element simulation, guided by experimental data, to understand the effect of water evaporation from the liquid meniscus at the tip of the nozzle for deposition of free-standing copper microwires in MCED process.

  13. Two-Step Multi-Physics Analysis of an Annular Linear Induction Pump for Fission Power Systems

    NASA Technical Reports Server (NTRS)

    Geng, Steven M.; Reid, Terry V.

    2016-01-01

    One of the key technologies associated with fission power systems (FPS) is the annular linear induction pump (ALIP). ALIPs are used to circulate liquid-metal fluid for transporting thermal energy from the nuclear reactor to the power conversion device. ALIPs designed and built to date for FPS project applications have not performed up to expectations. A unique, two-step approach was taken toward the multi-physics examination of an ALIP using ANSYS Maxwell 3D and Fluent. This multi-physics approach was developed so that engineers could investigate design variations that might improve pump performance. Of interest was to determine if simple geometric modifications could be made to the ALIP components with the goal of increasing the Lorentz forces acting on the liquid-metal fluid, which in turn would increase pumping capacity. The multi-physics model first calculates the Lorentz forces acting on the liquid metal fluid in the ALIP annulus. These forces are then used in a computational fluid dynamics simulation as (a) internal boundary conditions and (b) source functions in the momentum equations within the Navier-Stokes equations. The end result of the two-step analysis is a predicted pump pressure rise that can be compared with experimental data.

  14. Design and Analysis of a New Hair Sensor for Multi-Physical Signal Measurement

    PubMed Central

    Yang, Bo; Hu, Di; Wu, Lei

    2016-01-01

    A new hair sensor for multi-physical signal measurements, including acceleration, angular velocity and air flow, is presented in this paper. The entire structure consists of a hair post, a torsional frame and a resonant signal transducer. The hair post is utilized to sense and deliver the physical signals of the acceleration and the air flow rate. The physical signals are converted into frequency signals by the resonant transducer. The structure is optimized through finite element analysis. The simulation results demonstrate that the hair sensor has a frequency of 240 Hz in the first mode for the acceleration or the air flow sense, 3115 Hz in the third and fourth modes for the resonant conversion, and 3467 Hz in the fifth and sixth modes for the angular velocity transformation, respectively. All the above frequencies present in a reasonable modal distribution and are separated from interference modes. The input-output analysis of the new hair sensor demonstrates that the scale factor of the acceleration is 12.35 Hz/g, the scale factor of the angular velocity is 0.404 nm/deg/s and the sensitivity of the air flow is 1.075 Hz/(m/s)2, which verifies the multifunction sensitive characteristics of the hair sensor. Besides, the structural optimization of the hair post is used to improve the sensitivity of the air flow rate and the acceleration. The analysis results illustrate that the hollow circular hair post can increase the sensitivity of the air flow and the II-shape hair post can increase the sensitivity of the acceleration. Moreover, the thermal analysis confirms the scheme of the frequency difference for the resonant transducer can prominently eliminate the temperature influences on the measurement accuracy. The air flow analysis indicates that the surface area increase of hair post is significantly beneficial for the efficiency improvement of the signal transmission. In summary, the structure of the new hair sensor is proved to be feasible by comprehensive

  15. Supercomputing with TOUGH2 family codes for coupled multi-physics simulations of geologic carbon sequestration

    NASA Astrophysics Data System (ADS)

    Yamamoto, H.; Nakajima, K.; Zhang, K.; Nanai, S.

    2015-12-01

    scalabilities showing almost linear speedup against number of processors up to over ten thousand cores. Generally this allows us to perform coupled multi-physics (THC) simulations on high resolution geologic models with multi-million grid in a practical time (e.g., less than a second per time step).

  16. Design and Analysis of a New Hair Sensor for Multi-Physical Signal Measurement.

    PubMed

    Yang, Bo; Hu, Di; Wu, Lei

    2016-07-08

    A new hair sensor for multi-physical signal measurements, including acceleration, angular velocity and air flow, is presented in this paper. The entire structure consists of a hair post, a torsional frame and a resonant signal transducer. The hair post is utilized to sense and deliver the physical signals of the acceleration and the air flow rate. The physical signals are converted into frequency signals by the resonant transducer. The structure is optimized through finite element analysis. The simulation results demonstrate that the hair sensor has a frequency of 240 Hz in the first mode for the acceleration or the air flow sense, 3115 Hz in the third and fourth modes for the resonant conversion, and 3467 Hz in the fifth and sixth modes for the angular velocity transformation, respectively. All the above frequencies present in a reasonable modal distribution and are separated from interference modes. The input-output analysis of the new hair sensor demonstrates that the scale factor of the acceleration is 12.35 Hz/g, the scale factor of the angular velocity is 0.404 nm/deg/s and the sensitivity of the air flow is 1.075 Hz/(m/s)², which verifies the multifunction sensitive characteristics of the hair sensor. Besides, the structural optimization of the hair post is used to improve the sensitivity of the air flow rate and the acceleration. The analysis results illustrate that the hollow circular hair post can increase the sensitivity of the air flow and the II-shape hair post can increase the sensitivity of the acceleration. Moreover, the thermal analysis confirms the scheme of the frequency difference for the resonant transducer can prominently eliminate the temperature influences on the measurement accuracy. The air flow analysis indicates that the surface area increase of hair post is significantly beneficial for the efficiency improvement of the signal transmission. In summary, the structure of the new hair sensor is proved to be feasible by comprehensive

  17. Gene expression and cAMP.

    PubMed Central

    Nagamine, Y; Reich, E

    1985-01-01

    By comparing the 5'-flanking region of the porcine gene for the urokinase form of plasminogen activator with those of other cAMP-regulated genes, we identify a 29-nucleotide sequence that is tentatively proposed as the cAMP-regulatory unit. Homologous sequences are present (i) in the cAMP-regulated rat tyrosine aminotransferase, prolactin, and phosphoenolpyruvate carboxykinase genes and (ii) 5' to the transcription initiation sites of cAMP-regulated Escherichia coli genes. From this we conclude that the expression of cAMP-responsive genes in higher eukaryotes may be controlled, as in E. coli, by proteins that form complexes with cAMP and then show sequence-specific DNA-binding properties. The complex formed by cAMP and the regulatory subunit of the type II mammalian protein kinase might be one candidate for this function. Based on several homologies we suggest that this subunit may have retained both the DNA-binding specificity and transcription-regulating properties in addition to the nucleotide-binding domains of the bacterial cAMP-binding protein. If this were so, dissociation of protein kinase by cAMP would activate two processes: (i) protein phosphorylation by the catalytic subunit and (ii) transcription regulation by the regulatory subunit. PMID:2991882

  18. Experimental Characterisation and Multi-Physic Modelling of Direct Bonding Mechanical Behaviour: Application to Spatial Optical Systems

    NASA Astrophysics Data System (ADS)

    Cocheteau, N.; Maurel-Pantel, A.; Lebon, F.; Rosu, I.; Ait-Zaid, S.; Savin de Larclause, I.; Salaun, Y.

    2014-06-01

    Direct bonding is a well-known process. However in order to use this process in spatial instrument fabrication the mechanical resistance needs to be quantified precisely. In order to improve bonded strength, optimal parameters of the process are found by studying the influence of annealing time, temperature and roughness which are studied using three experimental methods: double shear, cleavage and wedge tests. Those parameters are chosen thanks to the appearance of time/temperature equivalence. All results brought out the implementation of a multi-physic model to predict the mechanical behavior of direct bonding interface.

  19. Targeting protein-protein interactions within the cyclic AMP signaling system as a therapeutic strategy for cardiovascular disease.

    PubMed

    Lee, Louisa C Y; Maurice, Donald H; Baillie, George S

    2013-03-01

    The cAMP signaling system can trigger precise physiological cellular responses that depend on the fidelity of many protein-protein interactions, which act to bring together signaling intermediates at defined locations within cells. In the heart, cAMP participates in the fine control of excitation-contraction coupling, hence, any disregulation of this signaling cascade can lead to cardiac disease. Due to the ubiquitous nature of the cAMP pathway, general inhibitors of cAMP signaling proteins such as PKA, EPAC and PDEs would act non-specifically and universally, increasing the likelihood of serious 'off target' effects. Recent advances in the discovery of peptides and small molecules that disrupt the protein-protein interactions that underpin cellular targeting of cAMP signaling proteins are described and discussed.

  20. Three Yersinia enterocolitica AmpD Homologs Participate in the Multi-Step Regulation of Chromosomal Cephalosporinase, AmpC

    PubMed Central

    Liu, Chang; Wang, Xin; Chen, Yuhuang; Hao, Huijing; Li, Xu; Liang, Junrong; Duan, Ran; Li, Chuchu; Zhang, Jing; Shao, Shihe; Jing, Huaiqi

    2016-01-01

    In many gram negative bacilli, AmpD plays a key role in both cell well-recycling pathway and β-lactamase regulation, inactivation of the ampD causes the accumulation of 1,6-anhydromuropeptides, and results in the ampC overproduction. In Yersinia enterocolitica, the regulation of ampC expression may also rely on the ampR-ampC system, the role of AmpD in this species is still unknown. In this study, three AmpD homologs (AmpD1, AmpD2, and AmpD3) have been identified in complete sequence of strain Y. enterocolitica subsp. palearctica 105.5R(r). To understand the role of three AmpD homologs, several mutant strains were constructed and analyzed where a rare ampC regulation mechanism was observed: low-effective ampD2 and ampD3 cooperate with the high-effective ampD1 in the three levels regulation of ampC expression. Enterobacteriaceae was used to be supposed to regulate ampC expression by two steps, three steps regulation was only observed in Pseudomonas aeruginosa. In this study, we first reported that Enterobacteriaceae Y. enterocolitica can also possess a three steps stepwise regulation mechanism, regulating the ampC expression precisely. PMID:27588018

  1. Three Yersinia enterocolitica AmpD Homologs Participate in the Multi-Step Regulation of Chromosomal Cephalosporinase, AmpC.

    PubMed

    Liu, Chang; Wang, Xin; Chen, Yuhuang; Hao, Huijing; Li, Xu; Liang, Junrong; Duan, Ran; Li, Chuchu; Zhang, Jing; Shao, Shihe; Jing, Huaiqi

    2016-01-01

    In many gram negative bacilli, AmpD plays a key role in both cell well-recycling pathway and β-lactamase regulation, inactivation of the ampD causes the accumulation of 1,6-anhydromuropeptides, and results in the ampC overproduction. In Yersinia enterocolitica, the regulation of ampC expression may also rely on the ampR-ampC system, the role of AmpD in this species is still unknown. In this study, three AmpD homologs (AmpD1, AmpD2, and AmpD3) have been identified in complete sequence of strain Y. enterocolitica subsp. palearctica 105.5R(r). To understand the role of three AmpD homologs, several mutant strains were constructed and analyzed where a rare ampC regulation mechanism was observed: low-effective ampD2 and ampD3 cooperate with the high-effective ampD1 in the three levels regulation of ampC expression. Enterobacteriaceae was used to be supposed to regulate ampC expression by two steps, three steps regulation was only observed in Pseudomonas aeruginosa. In this study, we first reported that Enterobacteriaceae Y. enterocolitica can also possess a three steps stepwise regulation mechanism, regulating the ampC expression precisely.

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

    SciTech Connect

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

    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.

  3. Multi-Physics Modelling of Fault Mechanics Using REDBACK: A Parallel Open-Source Simulator for Tightly Coupled Problems

    NASA Astrophysics Data System (ADS)

    Poulet, Thomas; Paesold, Martin; Veveakis, Manolis

    2017-03-01

    Faults play a major role in many economically and environmentally important geological systems, ranging from impermeable seals in petroleum reservoirs to fluid pathways in ore-forming hydrothermal systems. Their behavior is therefore widely studied and fault mechanics is particularly focused on the mechanisms explaining their transient evolution. Single faults can change in time from seals to open channels as they become seismically active and various models have recently been presented to explain the driving forces responsible for such transitions. A model of particular interest is the multi-physics oscillator of Alevizos et al. (J Geophys Res Solid Earth 119(6), 4558-4582, 2014) which extends the traditional rate and state friction approach to rate and temperature-dependent ductile rocks, and has been successfully applied to explain spatial features of exposed thrusts as well as temporal evolutions of current subduction zones. In this contribution we implement that model in REDBACK, a parallel open-source multi-physics simulator developed to solve such geological instabilities in three dimensions. The resolution of the underlying system of equations in a tightly coupled manner allows REDBACK to capture appropriately the various theoretical regimes of the system, including the periodic and non-periodic instabilities. REDBACK can then be used to simulate the drastic permeability evolution in time of such systems, where nominally impermeable faults can sporadically become fluid pathways, with permeability increases of several orders of magnitude.

  4. Transient multi-physics analysis of a magnetorheological shock absorber with the inverse Jiles-Atherton hysteresis model

    NASA Astrophysics Data System (ADS)

    Zheng, Jiajia; Li, Yancheng; Li, Zhaochun; Wang, Jiong

    2015-10-01

    This paper presents multi-physics modeling of an MR absorber considering the magnetic hysteresis to capture the nonlinear relationship between the applied current and the generated force under impact loading. The magnetic field, temperature field, and fluid dynamics are represented by the Maxwell equations, conjugate heat transfer equations, and Navier-Stokes equations. These fields are coupled through the apparent viscosity and the magnetic force, both of which in turn depend on the magnetic flux density and the temperature. Based on a parametric study, an inverse Jiles-Atherton hysteresis model is used and implemented for the magnetic field simulation. The temperature rise of the MR fluid in the annular gap caused by core loss (i.e. eddy current loss and hysteresis loss) and fluid motion is computed to investigate the current-force behavior. A group of impulsive tests was performed for the manufactured MR absorber with step exciting currents. The numerical and experimental results showed good agreement, which validates the effectiveness of the proposed multi-physics FEA model.

  5. A full-spectrum analysis of high-speed train interior noise under multi-physical-field coupling excitations

    NASA Astrophysics Data System (ADS)

    Zheng, Xu; Hao, Zhiyong; Wang, Xu; Mao, Jie

    2016-06-01

    High-speed-railway-train interior noise at low, medium, and high frequencies could be simulated by finite element analysis (FEA) or boundary element analysis (BEA), hybrid finite element analysis-statistical energy analysis (FEA-SEA) and statistical energy analysis (SEA), respectively. First, a new method named statistical acoustic energy flow (SAEF) is proposed, which can be applied to the full-spectrum HST interior noise simulation (including low, medium, and high frequencies) with only one model. In an SAEF model, the corresponding multi-physical-field coupling excitations are firstly fully considered and coupled to excite the interior noise. The interior noise attenuated by sound insulation panels of carriage is simulated through modeling the inflow acoustic energy from the exterior excitations into the interior acoustic cavities. Rigid multi-body dynamics, fast multi-pole BEA, and large-eddy simulation with indirect boundary element analysis are first employed to extract the multi-physical-field excitations, which include the wheel-rail interaction forces/secondary suspension forces, the wheel-rail rolling noise, and aerodynamic noise, respectively. All the peak values and their frequency bands of the simulated acoustic excitations are validated with those from the noise source identification test. Besides, the measured equipment noise inside equipment compartment is used as one of the excitation sources which contribute to the interior noise. Second, a full-trimmed FE carriage model is firstly constructed, and the simulated modal shapes and frequencies agree well with the measured ones, which has validated the global FE carriage model as well as the local FE models of the aluminum alloy-trim composite panel. Thus, the sound transmission loss model of any composite panel has indirectly been validated. Finally, the SAEF model of the carriage is constructed based on the accurate FE model and stimulated by the multi-physical-field excitations. The results show

  6. C++ Coding Standards for the AMP Project

    SciTech Connect

    Evans, Thomas M; Clarno, Kevin T

    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].

  7. AMP metabolism in the marine bacterium Beneckea natriegens.

    PubMed

    Pickard, M A; Whelihan, J A; Knowles, C J

    1980-05-01

    The catabolism of AMP by preparations from Beneckea natriegens has been reexamined. In the absence of ATP, cell-free extracts catabolized AMP via adenosine to inosine. When ATP was present, adenylate kinase converted AMP to ADP, lowering the rate of AMP catabolism. Particle-free supernatants (225,000 x g) metabolized AMP alone slowly, but adenylate kinase was active when ATP was added. Washed particulate fractions contained AMP nucleotidase activity which converted AMP to adenosine; in the presence of ATP, adenosine formation was reduced by residual adenylate kinase associated with the particulate fraction. IMP was not detected as a metabolite in these experiments.

  8. Investigation on Multi-Physics Simulation-Based Virtual Machining System for Vibratory Finishing of Integrally Bladed Rotors (IBRS)

    NASA Astrophysics Data System (ADS)

    Achiamah-Ampomah, N.; Cheng, Kai

    2016-02-01

    An investigation was carried out to improve the slow surface finishing times of integrally bladed rotors (IBRs) in the aerospace industry. Traditionally they are finished by hand, or more currently by abrasive flow machining. The use of a vibratory finishing technique to improve process times has been suggested; however as a largely empirical process, very few studies have been done to improve and optimize the cycle times, showing that critical and ongoing research is still needed in this area. An extensive review of the literature was carried out, and the findings used to identify the key parameters and model equations which govern the vibratory process. Recommendations were made towards a multi-physics-based simulation model, as well as projections made for the future of vibratory finishing and optimization of surface finishes and cycle times.

  9. Atmospheric, Magnetospheric and Plasmas in space (AMPS) spacelab payload definition study. Volume 4. Part 1, AMPS program specification

    NASA Technical Reports Server (NTRS)

    Keeley, J. T.

    1976-01-01

    The AMPS Program Specification delineates the AMPS Program requirements consistent with the resources defined in the AMPS Project Plan. All subsidiary specifications and requirements shall conform to the requirements presented. The requirements hierarchy for the AMPS program is illustrated. A brief description of each of the requirements documents and their intended use is provided.

  10. Multi-physics analysis for MEMS meshing micro-gear contacts

    NASA Astrophysics Data System (ADS)

    Theodossiades, S.; Teodorescu, M.; Rahnejat, H.

    2008-03-01

    Recent scientific and technical advances have enabled deposition of thin coating layers, such as silica or alumina (down to a few nano-meters). These have enabled the manufacture of complete micro-scale interconnected structures in the emerging micro-electromechanical systems (MEMS). Most MEMS contain a significant number of micro-size gears, transmitting small torques at fairly high speeds. The very small size of these elements implies that in most cases the inertial forces are insignificant, and therefore, they can withstand relative velocities and accelerations, which are prohibitive for their macro-scale counterparts. Although the methods of manufacture have been relatively successful, the established theories based on continuum mechanics do not immediately extend to some of the micro-scale interactions. Consequently, there is an element of empirical approach in their fabrication, which manifests itself in their inherent unreliability, particularly vis-à-vis tribological performance.

  11. Dual chemotaxis signalling regulates Dictyostelium development: intercellular cyclic AMP pulses and intracellular F-actin disassembly waves induce each other.

    PubMed

    Vicker, Michael G; Grutsch, James F

    2008-10-01

    Aggregating Dictyostelium discoideum amoebae periodically emit and relay cAMP, which regulates their chemotaxis and morphogenesis into a multicellular, differentiated organism. Cyclic AMP also stimulates F-actin assembly and chemotactic pseudopodium extension. We used actin-GFP expression to visualise for the first time intracellular F-actin assembly as a spatio-temporal indicator of cell reactions to cAMP, and thus the kinematics of cell communication, in aggregating streams. Every natural cAMP signal pulse induces an autowave of F-actin disassembly, which propagates from each cell's leading end to its trailing end at a linear rate, much slower than the calculated and measured velocities of cAMP diffusion in aggregating Dictyostelium. A sequence of transient reactions follows behind the wave, including anterior F-actin assembly, chemotactic pseudopodium extension and cell advance at the cell front and, at the back, F-actin assembly, extension of a small retrograde pseudopodium (forcing a brief cell retreat) and chemotactic stimulation of the following cell, yielding a 20s cAMP relay delay. These dynamics indicate that stream cell behaviour is mediated by a dual signalling system: a short-range cAMP pulse directed from one cell tail to an immediately following cell front and a slower, long-range wave of intracellular F-actin disassembly, each inducing the other.

  12. beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics.

    PubMed

    Violin, Jonathan D; DiPilato, Lisa M; Yildirim, Necmettin; Elston, Timothy C; Zhang, Jin; Lefkowitz, Robert J

    2008-02-01

    G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

  13. Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling.

    PubMed

    Dema, Alessandro; Perets, Ekaterina; Schulz, Maike Svenja; Deák, Veronika Anita; Klussmann, Enno

    2015-12-01

    The second messenger cyclic adenosine monophosphate (cAMP) can bind and activate protein kinase A (PKA). The cAMP/PKA system is ubiquitous and involved in a wide array of biological processes and therefore requires tight spatial and temporal regulation. Important components of the safeguard system are the A-kinase anchoring proteins (AKAPs), a heterogeneous family of scaffolding proteins defined by its ability to directly bind PKA. AKAPs tether PKA to specific subcellular compartments, and they bind further interaction partners to create local signalling hubs. The recent discovery of new AKAPs and advances in the field that shed light on the relevance of these hubs for human disease highlight unique opportunities for pharmacological modulation. This review exemplifies how interference with signalling, particularly cAMP signalling, at such hubs can reshape signalling responses and discusses how this could lead to novel pharmacological concepts for the treatment of disease with an unmet medical need such as cardiovascular disease and cancer.

  14. Multi-physics damage sensing in nano-engineered structural composites

    NASA Astrophysics Data System (ADS)

    Guzmán de Villoria, Roberto; Yamamoto, Namiko; Miravete, Antonio; Wardle, Brian L.

    2011-05-01

    Non-destructive evaluation techniques can offer viable diagnostic and prognostic routes to mitigating failures in engineered structures such as bridges, buildings and vehicles. However, existing techniques have significant drawbacks, including poor spatial resolution and limited in situ capabilities. We report here a novel approach where structural advanced composites containing electrically conductive aligned carbon nanotubes (CNTs) are ohmically heated via simple electrical contacts, and damage is visualized via thermographic imaging. Damage, in the form of cracks and other discontinuities, usefully increases resistance to both electrical and thermal transport in these materials, which enables tomographic full-field damage assessment in many cases. Characteristics of the technique include the ability for real-time measurement of the damage state during loading, low-power operation (e.g. 15 °C rise at 1 W), and beyond state-of-the-art spatial resolution for sensing damage in composites. The enhanced thermographic technique is a novel and practical approach for in situ monitoring to ascertain structural health and to prevent structural failures in engineered structures such as aerospace and automotive vehicles and wind turbine blades, among others.

  15. Multi-physics damage sensing in nano-engineered structural composites.

    PubMed

    de Villoria, Roberto Guzmán; Yamamoto, Namiko; Miravete, Antonio; Wardle, Brian L

    2011-05-06

    Non-destructive evaluation techniques can offer viable diagnostic and prognostic routes to mitigating failures in engineered structures such as bridges, buildings and vehicles. However, existing techniques have significant drawbacks, including poor spatial resolution and limited in situ capabilities. We report here a novel approach where structural advanced composites containing electrically conductive aligned carbon nanotubes (CNTs) are ohmically heated via simple electrical contacts, and damage is visualized via thermographic imaging. Damage, in the form of cracks and other discontinuities, usefully increases resistance to both electrical and thermal transport in these materials, which enables tomographic full-field damage assessment in many cases. Characteristics of the technique include the ability for real-time measurement of the damage state during loading, low-power operation (e.g. 15 °C rise at 1 W), and beyond state-of-the-art spatial resolution for sensing damage in composites. The enhanced thermographic technique is a novel and practical approach for in situ monitoring to ascertain structural health and to prevent structural failures in engineered structures such as aerospace and automotive vehicles and wind turbine blades, among others.

  16. Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds.

    PubMed

    Yokoyama, Seiya; Kato, Kouji; Koba, Atsuko; Minami, Yuji; Watanabe, Keiichi; Yagi, Fumio

    2008-12-01

    Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

  17. cAMP and EPAC Signaling Functionally Replace OCT4 During Induced Pluripotent Stem Cell Reprogramming.

    PubMed

    Fritz, Ashley L; Adil, Maroof M; Mao, Sunnie R; Schaffer, David V

    2015-05-01

    The advent of induced pluripotent stem cells--generated via the ectopic overexpression of reprogramming factors such as OCT4, SOX2, KLF4, and C-MYC (OSKM) in a differentiated cell type--has enabled groundbreaking research efforts in regenerative medicine, disease modeling, and drug discovery. Although initial studies have focused on the roles of nuclear factors, increasing evidence highlights the importance of signal transduction during reprogramming. By utilizing a quantitative, medium-throughput screen to initially identify signaling pathways that could potentially replace individual transcription factors during reprogramming, we initially found that several pathways--such as Notch, Smoothened, and cyclic AMP (cAMP) signaling--were capable of generating alkaline phosphatase positive colonies in the absence of OCT4, the most stringently required Yamanaka factor. After further investigation, we discovered that cAMP signal activation could functionally replace OCT4 to induce pluripotency, and results indicate that the downstream exchange protein directly activated by cAMP (EPAC) signaling pathway rather than protein kinase A (PKA) signaling is necessary and sufficient for this function. cAMP signaling may reduce barriers to reprogramming by contributing to downstream epithelial gene expression, decreasing mesenchymal gene expression, and increasing proliferation. Ultimately, these results elucidate mechanisms that could lead to new reprogramming methodologies and advance our understanding of stem cell biology.

  18. A hierarchical multi-physics model for design of high toughness steels

    NASA Astrophysics Data System (ADS)

    Hao, Su; Moran, Brian; Kam Liu, Wing; Olson, Gregory B.

    2003-05-01

    In support of the computational design of high toughness steels as hierarchically structured materials, a multiscale, multiphysics methodology is developed for a `ductile fracture simulator.' At the nanometer scale, the method unites continuum mechanics with quantum physics, using first-principles calculations to predict the force-distance laws for interfacial separation with both normal and plastic sliding components. The predicted adhesion behavior is applied to the description of interfacial decohesion for both micron-scale primary inclusions governing primary void formation and submicron-scale secondary particles governing microvoid-based shear localization that accelerates primary void coalescence. Fine scale deformation is described by a `Particle Dynamics' method that extends the framework of molecular dynamics to multi-atom aggregates. This is combined with other meshfree and finite-element methods in two-level cell modeling to provide a hierarchical constitutive model for crack advance, combining conventional plasticity, microstructural damage, strain gradient effects and transformation plasticity from dispersed metastable austenite. Detailed results of a parallel experimental study of a commercial steel are used to calibrate the model at multiple scales. An initial application provides a Toughness-Strength-Adhesion diagram defining the relation among alloy strength, inclusion adhesion energy and fracture toughness as an aid to microstructural design. The analysis of this paper introduces an approach of creative steel design that can be stated as the exploration of the effective connections among the five key-components: elements selection, process design, micro/nanostructure optimization, desirable properties and industrial performance by virtue of innovations and inventions.

  19. Modeling and simulation of multi-physics multi-scale transport phenomenain bio-medical applications

    NASA Astrophysics Data System (ADS)

    Kenjereš, Saša

    2014-08-01

    We present a short overview of some of our most recent work that combines the mathematical modeling, advanced computer simulations and state-of-the-art experimental techniques of physical transport phenomena in various bio-medical applications. In the first example, we tackle predictions of complex blood flow patterns in the patient-specific vascular system (carotid artery bifurcation) and transfer of the so-called "bad" cholesterol (low-density lipoprotein, LDL) within the multi-layered artery wall. This two-way coupling between the blood flow and corresponding mass transfer of LDL within the artery wall is essential for predictions of regions where atherosclerosis can develop. It is demonstrated that a recently developed mathematical model, which takes into account the complex multi-layer arterial-wall structure, produced LDL profiles within the artery wall in good agreement with in-vivo experiments in rabbits, and it can be used for predictions of locations where the initial stage of development of atherosclerosis may take place. The second example includes a combination of pulsating blood flow and medical drug delivery and deposition controlled by external magnetic field gradients in the patient specific carotid artery bifurcation. The results of numerical simulations are compared with own PIV (Particle Image Velocimetry) and MRI (Magnetic Resonance Imaging) in the PDMS (silicon-based organic polymer) phantom. A very good agreement between simulations and experiments is obtained for different stages of the pulsating cycle. Application of the magnetic drug targeting resulted in an increase of up to ten fold in the efficiency of local deposition of the medical drug at desired locations. Finally, the LES (Large Eddy Simulation) of the aerosol distribution within the human respiratory system that includes up to eight bronchial generations is performed. A very good agreement between simulations and MRV (Magnetic Resonance Velocimetry) measurements is obtained

  20. The atmospheric component of the Mediterranean Sea water budget in a WRF multi-physics ensemble and observations

    NASA Astrophysics Data System (ADS)

    Di Luca, Alejandro; Flaounas, Emmanouil; Drobinski, Philippe; Brossier, Cindy Lebeaupin

    2014-11-01

    The use of high resolution atmosphere-ocean coupled regional climate models to study possible future climate changes in the Mediterranean Sea requires an accurate simulation of the atmospheric component of the water budget (i.e., evaporation, precipitation and runoff). A specific configuration of the version 3.1 of the weather research and forecasting (WRF) regional climate model was shown to systematically overestimate the Mediterranean Sea water budget mainly due to an excess of evaporation (~1,450 mm yr-1) compared with observed estimations (~1,150 mm yr-1). In this article, a 70-member multi-physics ensemble is used to try to understand the relative importance of various sub-grid scale processes in the Mediterranean Sea water budget and to evaluate its representation by comparing simulated results with observed-based estimates. The physics ensemble was constructed by performing 70 1-year long simulations using version 3.3 of the WRF model by combining six cumulus, four surface/planetary boundary layer and three radiation schemes. Results show that evaporation variability across the multi-physics ensemble (˜10 % of the mean evaporation) is dominated by the choice of the surface layer scheme that explains more than ˜70 % of the total variance and that the overestimation of evaporation in WRF simulations is generally related with an overestimation of surface exchange coefficients due to too large values of the surface roughness parameter and/or the simulation of too unstable surface conditions. Although the influence of radiation schemes on evaporation variability is small (˜13 % of the total variance), radiation schemes strongly influence exchange coefficients and vertical humidity gradients near the surface due to modifications of temperature lapse rates. The precipitation variability across the physics ensemble (˜35 % of the mean precipitation) is dominated by the choice of both cumulus (˜55 % of the total variance) and planetary boundary layer (˜32 % of

  1. Effect of some new cAMP analogs on cAMP-dependent protein kinase isoenzymes.

    PubMed

    Szücs, K; Sági, G; Vereb, G

    1992-06-01

    1. Ten new cAMP analogs were synthesized by replacing the purine ring with with indazole, benzimidazole or benztriazole and/or their nitro and amino derivatives. 2. Each analog proved effective in activating cAMP-dependent protein kinase I (PK-I) purified from rabbit skeletal muscle and cAMP-dependent protein kinase II (PK-II) from bovine heart and chasing 8-[3H]cAMP bound to regulatory subunits in the half-maximal effective concentrations of 2 x 10(-8)-8 x 10(-6) M. 3. The N-1-beta-D-ribofuranosyl-indazole-3'5'-cyclophosphate(I) proved a very poor chaser and activator of both isoenzymes, but when indazole was attached at its N-2 to ribose (IV) or when its H at C-4 (equivalent to the position of amino-group in adenine) was substituted by an amino-(III) or especially nitro-group (II) its efficiency was dramatically increased. 4. Analogs containing benztriazole ring proved as powerful as cAMP irrespective of the presence of substituents (VII-X). 5. Benzimidazole derivatives with amino-(VI) or nitro-group (V) activated PK-II 3 and 20 times better than PK-I. 6. Attaching of ribose to N-2 of indazole or benztriazole increased the affinity to PK-II 10 and 4 times, respectively. 7. Chasing efficiency of cAMP analogs at half-saturating [3H]cAMP tended to correlate with activating potency only for PK-I but at saturating [3H]cAMP concentration for both isoenzymes. 8. On the basis of synergistic activation with 8-Br-cAMP a site 2-selective binding of nitro-benzimidazole (V) and unsubstituted benztriazole (VII) derivatives to PK-II is suggested.

  2. Escherichia coli exports cyclic AMP via TolC.

    PubMed

    Hantke, Klaus; Winkler, Karin; Schultz, Joachim E

    2011-03-01

    In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

  3. Numerical simulation and experimental validation of biofilm in a multi-physics framework using an SPH based method

    NASA Astrophysics Data System (ADS)

    Soleimani, Meisam; Wriggers, Peter; Rath, Henryke; Stiesch, Meike

    2016-10-01

    In this paper, a 3D computational model has been developed to investigate biofilms in a multi-physics framework using smoothed particle hydrodynamics (SPH) based on a continuum approach. Biofilm formation is a complex process in the sense that several physical phenomena are coupled and consequently different time-scales are involved. On one hand, biofilm growth is driven by biological reaction and nutrient diffusion and on the other hand, it is influenced by fluid flow causing biofilm deformation and interface erosion in the context of fluid and deformable solid interaction. The geometrical and numerical complexity arising from these phenomena poses serious complications and challenges in grid-based techniques such as finite element. Here the solution is based on SPH as one of the powerful meshless methods. SPH based computational modeling is quite new in the biological community and the method is uniquely robust in capturing the interface-related processes of biofilm formation such as erosion. The obtained results show a good agreement with experimental and published data which demonstrates that the model is capable of simulating and predicting overall spatial and temporal evolution of biofilm.

  4. A multi-physics study of Li-ion battery material Li1+xTi2O4

    NASA Astrophysics Data System (ADS)

    Jiang, Tonghu; Falk, Michael; Siva Shankar Rudraraju, Krishna; Garikipati, Krishna; van der Ven, Anton

    2013-03-01

    Recently, lithium ion batteries have been subject to intense scientific study due to growing demand arising from their utilization in portable electronics, electric vehicles and other applications. Most cathode materials in lithium ion batteries involve a two-phase process during charging and discharging, and the rate of these processes is typically limited by the slow interface mobility. We have undertaken modeling regarding how lithium diffusion in the interface region affects the motion of the phase boundary. We have developed a multi-physics computational method suitable for predicting time evolution of the driven interface. In this method, we calculate formation energies and migration energy barriers by ab initio methods, which are then approximated by cluster expansions. Monte Carlo calculation is further employed to obtain thermodynamic and kinetic information, e.g., anisotropic interfacial energies, and mobilities, which are used to parameterize continuum modeling of the charging and discharging processes. We test this methodology on spinel Li1+xTi2O4. Elastic effects are incorporated into the calculations to determine the effect of variations in modulus and strain on stress concentrations and failure modes within the material. We acknowledge support by the National Science Foundation Cyber Discovery and Innovation Program under Award No. 1027765.

  5. Parallel Monte Carlo transport modeling in the context of a time-dependent, three-dimensional multi-physics code

    SciTech Connect

    Procassini, R.J.

    1997-12-31

    The fine-scale, multi-space resolution that is envisioned for accurate simulations of complex weapons systems in three spatial dimensions implies flop-rate and memory-storage requirements that will only be obtained in the near future through the use of parallel computational techniques. Since the Monte Carlo transport models in these simulations usually stress both of these computational resources, they are prime candidates for parallelization. The MONACO Monte Carlo transport package, which is currently under development at LLNL, will utilize two types of parallelism within the context of a multi-physics design code: decomposition of the spatial domain across processors (spatial parallelism) and distribution of particles in a given spatial subdomain across additional processors (particle parallelism). This implementation of the package will utilize explicit data communication between domains (message passing). Such a parallel implementation of a Monte Carlo transport model will result in non-deterministic communication patterns. The communication of particles between subdomains during a Monte Carlo time step may require a significant level of effort to achieve a high parallel efficiency.

  6. Variable Resonance Frequency Selection for Fishbone-Shaped Microelectromechanical System Resonator Based on Multi-Physics Simulation

    NASA Astrophysics Data System (ADS)

    Kuroda, Shinjiro; Suzuki, Naoya; Tanigawa, Hiroshi; Suzuki, Kenichiro

    2013-06-01

    In this paper, we present and demonstrate the principle of variable resonance frequency selection by using a fishbone-shaped microelectromechanical system (MEMS) resonator. To analyze resonator displacement caused by an electrostatic force, a multi-physics simulation, which links the applied voltage load to the mechanical domain, is carried out. The simulation clearly shows that resonators are operated by three kinds of electrostatic force exerted on the beam. A new frequency selection algorithm that selects only one among various resonant modes is also presented. The conversion matrix that transforms the voltages applied to each driving electrode into the resonant beam displacement at each resonant mode is first derived by experimental measurements. Following this, the matrix is used to calculate a set of voltages for maximizing the rejection ratio in each resonant mode. This frequency selection method is applied in a fishbone-shaped MEMS resonator with five driving electrodes and the frequency selection among the 1st resonant mode to the 5th resonant mode is successfully demonstrated. From a fine adjustment of the voltage set, a 42 dB rejection ratio is obtained.

  7. Multi-Physics Modeling of Molten Salt Transport in Solid Oxide Membrane (SOM) Electrolysis and Recycling of Magnesium

    SciTech Connect

    Powell, Adam; Pati, Soobhankar

    2012-03-11

    Solid Oxide Membrane (SOM) Electrolysis is a new energy-efficient zero-emissions process for producing high-purity magnesium and high-purity oxygen directly from industrial-grade MgO. SOM Recycling combines SOM electrolysis with electrorefining, continuously and efficiently producing high-purity magnesium from low-purity partially oxidized scrap. In both processes, electrolysis and/or electrorefining take place in the crucible, where raw material is continuously fed into the molten salt electrolyte, producing magnesium vapor at the cathode and oxygen at the inert anode inside the SOM. This paper describes a three-dimensional multi-physics finite-element model of ionic current, fluid flow driven by argon bubbling and thermal buoyancy, and heat and mass transport in the crucible. The model predicts the effects of stirring on the anode boundary layer and its time scale of formation, and the effect of natural convection at the outer wall. MOxST has developed this model as a tool for scale-up design of these closely-related processes.

  8. AMPS/PC - AUTOMATIC MANUFACTURING PROGRAMMING SYSTEM

    NASA Technical Reports Server (NTRS)

    Schroer, B. J.

    1994-01-01

    The AMPS/PC system is a simulation tool designed to aid the user in defining the specifications of a manufacturing environment and then automatically writing code for the target simulation language, GPSS/PC. The domain of problems that AMPS/PC can simulate are manufacturing assembly lines with subassembly lines and manufacturing cells. The user defines the problem domain by responding to the questions from the interface program. Based on the responses, the interface program creates an internal problem specification file. This file includes the manufacturing process network flow and the attributes for all stations, cells, and stock points. AMPS then uses the problem specification file as input for the automatic code generator program to produce a simulation program in the target language GPSS. The output of the generator program is the source code of the corresponding GPSS/PC simulation program. The system runs entirely on an IBM PC running PC DOS Version 2.0 or higher and is written in Turbo Pascal Version 4 requiring 640K memory and one 360K disk drive. To execute the GPSS program, the PC must have resident the GPSS/PC System Version 2.0 from Minuteman Software. The AMPS/PC program was developed in 1988.

  9. AMPS Supporting Research and Technology (SR and T) report. Atmospheric, Magnetospheric and Plasmas in Space (AMPS) definition study

    NASA Technical Reports Server (NTRS)

    1976-01-01

    A listing of candidate technology areas that require additional study is presented. These candidate tasks, identified during the AMPS Phase B studies, are requisites to the design, development, and operation of the AMPS concept selected for preliminary design.

  10. Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein

    PubMed Central

    Underwood, Adam J.; Zhang, Yang; Metzger, Dennis W.; Bai, Guangchun

    2014-01-01

    Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling. PMID:25239824

  11. The cyclic AMP pathway is a sex-specific modifier of glioma risk in type 1 neurofibromatosis patients

    PubMed Central

    Warrington, Nicole M.; Sun, Tao; Luo, Jingqin; McKinstry, Robert C.; Parkin, Patricia C.; Ganzhorn, Sara; Spoljaric, Debra; Albers, Anne C.; Merkelson, Amanda; Stewart, Douglas R.; Stevenson, David A.; Viskochil, David; Druley, Todd E.; Forys, Jason T; Reilly, Karlyne M.; Fisher, Michael J.; Tabori, Uri; Allen, Jeffrey C.; Schiffman, Joshua D.; Gutmann, David H.; Rubin, Joshua B.

    2014-01-01

    Identifying modifiers of glioma risk in patients with type 1 neurofibromatosis (NF1) could help support personalized tumor surveillance, advance understanding of gliomagenesis and potentially identify novel therapeutic targets. Here we report genetic polymorphisms in the human adenylate cyclase gene ADCY8 which correlate with glioma risk in NF1 in a sex-specific manner, elevating risk in females while reducing risk in males. This finding extends earlier evidence of a role for cAMP in gliomagenesis based on results in a genetically engineered mouse model (Nf1 GEM). Thus, sexually dimorphic cAMP signaling might render males and females differentially sensitive to variation in cAMP levels. Using male and female Nf1 GEM, we found significant sex differences exist in cAMP regulation and in the growth promoting effects of cAMP suppression. Overall, our results establish a sex-specific role for cAMP regulation in human gliomagenesis, specifically identifying ADCY8 as a modifier of glioma risk in NF1. PMID:25381154

  12. Multi-Scale Multi-physics Methods Development for the Calculation of Hot-Spots in the NGNP

    SciTech Connect

    Downar, Thomas; Seker, Volkan

    2013-04-30

    Radioactive gaseous fission products are released out of the fuel element at a significantly higher rate when the fuel temperature exceeds 1600°C in high-temperature gas-cooled reactors (HTGRs). Therefore, it is of paramount importance to accurately predict the peak fuel temperature during all operational and design-basis accident conditions. The current methods used to predict the peak fuel temperature in HTGRs, such as the Next-Generation Nuclear Plant (NGNP), estimate the average fuel temperature in a computational mesh modeling hundreds of fuel pebbles or a fuel assembly in a pebble-bed reactor (PBR) or prismatic block type reactor (PMR), respectively. Experiments conducted in operating HTGRs indicate considerable uncertainty in the current methods and correlations used to predict actual temperatures. The objective of this project is to improve the accuracy in the prediction of local "hot" spots by developing multi-scale, multi-physics methods and implementing them within the framework of established codes used for NGNP analysis.The multi-scale approach which this project will implement begins with defining suitable scales for a physical and mathematical model and then deriving and applying the appropriate boundary conditions between scales. The macro scale is the greatest length that describes the entire reactor, whereas the meso scale models only a fuel block in a prismatic reactor and ten to hundreds of pebbles in a pebble bed reactor. The smallest scale is the micro scale--the level of a fuel kernel of the pebble in a PBR and fuel compact in a PMR--which needs to be resolved in order to calculate the peak temperature in a fuel kernel.

  13. The therapeutic applications of antimicrobial peptides (AMPs): a patent review.

    PubMed

    Kang, Hee-Kyoung; Kim, Cheolmin; Seo, Chang Ho; Park, Yoonkyung

    2017-01-01

    Antimicrobial peptides (AMPs) are small molecules with a broad spectrum of antibiotic activities against bacteria, yeasts, fungi, and viruses and cytotoxic activity on cancer cells, in addition to anti-inflammatory and immunomodulatory activities. Therefore, AMPs have garnered interest as novel therapeutic agents. Because of the rapid increase in drug-resistant pathogenic microorganisms, AMPs from synthetic and natural sources have been developed using alternative antimicrobial strategies. This article presents a broad analysis of patents referring to the therapeutic applications of AMPs since 2009. The review focuses on the universal trends in the effective design, mechanism, and biological evolution of AMPs.

  14. Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917.

    PubMed

    Yan, Huihui; Bao, Feifei; Zhao, Liping; Yu, Yanying; Tang, Jiaqin; Zhou, Xianxuan

    2015-11-01

    Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. (1)H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

  15. Active Materials for Photonic Systems (AMPS)

    DTIC Science & Technology

    2007-11-02

    market . Overall Program Summary The overall objective of the Active Materials for Photonic Systems (AMPS) program was to develop and demonstrate...mode fiber, with alignment tolerances of several microns functions well for data communications , single mode fiber is required for several significant...in the laser/optics community . Boeing and MCNC have signed a memorandum of agreement for commercialization and are actively seeking partners for

  16. New perspectives in cyclic AMP-mediated axon growth and guidance: The emerging epoch of Epac.

    PubMed

    Peace, Andrew G; Shewan, Derryck A

    2011-03-10

    In the search for a cure to brain and spinal cord injury much has been learned about the inhibitory environment of the central nervous system (CNS), and yet a clinical therapy remains elusive. In recent years great advances have been made in understanding intracellular molecular mechanisms that transduce cell surface receptor-mediated signals that neurons receive from their environment. Many of these signalling pathways share common mechanisms, which presents the possibility that manipulating activities of key cell signalling molecules such as those regulated by 3'-5'-cyclic adenosine monophosphate (cAMP) might allow axons to simultaneously overcome the inhibitory effects of a number of extracellular ligands. The identification of Epac, a novel direct intracellular target for cAMP, has opened up a new avenue of research that is beginning to explain how cAMP can mediate a range of neuronal functions including distinct axon growth and guidance decisions. With current research tools that allow more specific activation of proteins or knock-down of their expression, as well as quantitation of protein activities in live cells, it is already becoming clear that Epac plays highly important roles in the development and function of the nervous system. Here, we focus on emerging evidence that Epac mediates cAMP-regulated axon growth and chemoattraction, and thus represents a novel target for overcoming axon growth inhibition and promoting CNS regeneration.

  17. Cyclic AMP (cAMP) confers drug resistance against DNA damaging agents via PKAIA in CML cells.

    PubMed

    Xiao, Ling-Yi; Kan, Wai-Ming

    2017-01-05

    Cyclic adenosine monophosphate (cAMP) regulates many vital functions such as metabolism, proliferation, differentiation and death. Depending on cell types and stimulators, cAMP could either promote or attenuate cell death. cAMP signal can be transduced by protein kinase A (PKA) and/or exchange protein directly activated by cAMP (EPAC). In CML cells, cAMP may suppress their proliferation and enhance their differentiation. However, the role of cAMP on DNA damaging agent toxicity and the mechanism involved has not been studied. In this study, we studied the effect of cAMP on the sensitivity of CML cells to DNA damaging agents. We observed that forskolin (FSK) and dibutyryl-cAMP (DBcAMP) decreased cisplatin and etoposide-induced cell death in K562 cells. Moreover, PKA activator prevented K562 cells from DNA damaging agent-induced cell death while EPAC activator had no effect. Furthermore, we found that the PKA subtype, PKAIA, was involved in cAMP-attenuated resistance in K562 cells. Taken together, our results suggest that increased cAMP level confers CML cells to acquire a novel mechanism against DNA damaging agent toxicity via PKAIA. Thus, PKAIA inhibitor may be helpful in overcoming the resistance to DNA damaging agents in CML cells.

  18. Copper Regulates Cyclic AMP-Dependent Lipolysis

    PubMed Central

    Krishnamoorthy, Lakshmi; Cotruvo, Joseph A.; Chan, Jefferson; Kaluarachchi, Harini; Muchenditsi, Abigael; Pendyala, Venkata S.; Jia, Shang; Aron, Allegra T.; Ackerman, Cheri M.; Vander Wal, Mark N.; Guan, Timothy; Smaga, Lukas P.; Farhi, Samouil L.; New, Elizabeth J.; Lutsenko, Svetlana; Chang, Christopher J.

    2016-01-01

    Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium, and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining the body's weight and energy stores. Utilizing a murine model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we demonstrate that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue within a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype. PMID:27272565

  19. The Applied Mathematics for Power Systems (AMPS)

    SciTech Connect

    Chertkov, Michael

    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.

  20. Phospholipid metabolism in zymosan stimulated human monocytes: modulation by cyclic AMP (cAMP)

    SciTech Connect

    Godfrey R.W.; Manzi, R.M.; Hoffstein, S.T.

    1986-05-01

    Oxygenated products of arachidonic acid (AA) are critical components in the development of the inflammatory response. Monocytes exposed to inflammatory stimuli are capable of converting free AA into these bioactive molecules. However, the limiting step in the formation of these compounds is thought to be the mechanism responsible for the release of esterified AA from phospholipids. When (/sup 3/H) AA labeled monocytes were challenged with opsonized zymosan, 28 +/- 2% of the incorporated counts were released compared to 8 +/- 1% for the control. Upon pretreatment with isobutyl methyl xanthine (IBMX) or dibutyrl cyclic AMP (d-cAMP) zymosan stimulated AA release was markedly reduced. The IC/sub 50/'s were 4 x 10/sup -4/M and 7 x 10/sup -4/M respectively. Analysis of (/sup 3/H) AA incorporation into cellular phospholipids showed that phosphatidylcholine (PC) and phosphatidylinositol (PI) were the primary pools labeled. Loss of label from both of these pools was evident after exposure to zymosan, however, pretreatment of cells with IBMX or d-cAMP inhibited release of (/sup 3/H)AA from the PC pool but not from the PI pool. The results show that human monocytes challenged with opsonized zymosan release arachidonic acid via cAMP-dependent and independent pathways. Furthermore, they suggest that a phospholipase activity (possibly A/sub 2/) against PC is modulated by cAMP.

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

    PubMed

    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. Cyclic AMP induces transforming growth factor beta 2 gene expression and growth arrest in the human androgen-independent prostate carcinoma cell line PC-3.

    PubMed Central

    Bang, Y J; Kim, S J; Danielpour, D; O'Reilly, M A; Kim, K Y; Myers, C E; Trepel, J B

    1992-01-01

    The standard therapy for advanced prostate cancer is androgen ablation. Despite transitory responses, hormonally treated patients ultimately relapse with androgen-independent disease that is resistant to further hormonal manipulation and cytotoxic chemotherapy. To develop an additional approach to the treatment of advanced prostate cancer, we have been studying the signal transductions controlling the growth of human androgen-independent prostate carcinoma cell lines. We report here that elevation of intracellular cAMP markedly inhibits the growth of the hormone-refractory cell line PC-3. To examine the mechanism of cAMP action in PC-3 cells, we tested the effect of the cAMP analog dibutyryl cAMP (Bt2-cAMP) on the regulation of the potent negative growth factor transforming growth factor beta (TGF-beta). Bt2-cAMP selectively induced the secretion of TGF-beta 2 and not TGF-beta 1 by PC-3 cells. This TGF-beta 2 was shown to be bioactive by using the CCL-64 mink lung cell assay. TGF-beta 1 was not activated despite being present at 3-fold higher concentrations than TGF-beta 2. Northern analysis showed that Bt2-cAMP induced an increase in the five characteristic TGF-beta 2 transcripts and had no effect on the level of TGF-beta 1 or TGF-beta 3 transcripts. TGF-beta 2 induction was only weakly enhanced by cycloheximide and was completely inhibited by actinomycin D. These data show that Bt2-cAMP induces the expression of active TGF-beta 2 by PC-3 prostate carcinoma cells, suggesting a new approach to the treatment of prostate cancer and a new molecular mechanism of cAMP action. Images PMID:1373503

  3. The regulatory repertoire of Pseudomonas aeruginosa AmpC ß-lactamase regulator AmpR includes virulence genes.

    PubMed

    Balasubramanian, Deepak; Schneper, Lisa; Merighi, Massimo; Smith, Roger; Narasimhan, Giri; Lory, Stephen; Mathee, Kalai

    2012-01-01

    In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the

  4. The Regulatory Repertoire of Pseudomonas aeruginosa AmpC ß-Lactamase Regulator AmpR Includes Virulence Genes

    PubMed Central

    Balasubramanian, Deepak; Schneper, Lisa; Merighi, Massimo; Smith, Roger; Narasimhan, Giri; Lory, Stephen; Mathee, Kalai

    2012-01-01

    In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. In addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, we compared the transcriptional profile generated using DNA microarrays of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought, with the deletion of ampR influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Other virulence mechanisms including biofilm formation and QS-regulated acute virulence factors are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the microarray data. Further, using a Caenorhabditis elegans model, we demonstrate that a functional AmpR is required for P. aeruginosa pathogenicity. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. Further, we show differential regulation of other transcriptional regulators and sigma factors by AmpR, accounting for the extensive AmpR regulon. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating biofilm formation, a chronic infection phenotype. Unraveling this complex regulatory circuit will provide a better understanding of the bacterial response to antibiotics and how the

  5. Analysis of the PDFs of temperature from a multi-physics ensemble of climate change projections over the Iberian Peninsula

    NASA Astrophysics Data System (ADS)

    Jerez, Sonia; Montavez, Juan P.; Gomez-Navarro, Juan J.; Jimenez-Guerrero, Pedro; Lorente, Raquel; Garcia-Valero, Juan A.; Jimenez, Pedro A.; Gonzalez-Rouco, Jose F.; Zorita, Eduardo

    2010-05-01

    Regional climate change projections are affected by several sources of uncertainty. Some of them come from Global Circulation Models and scenarios.; others come from the downscaling process. In the case of dynamical downscaling, mainly using Regional Climate Models (RCM), the sources of uncertainty may involve nesting strategies, related to the domain position and resolution, soil characterization, internal variability, methods of solving the equations, and the configuration of model physics. Therefore, a probabilistic approach seems to be recommendable when projecting regional climate change. This problem is usually faced by performing an ensemble of simulations. The aim of this study is to evaluate the range of uncertainty in regional climate projections associated to changing the physical configuration in a RCM (MM5) as well as the capability when reproducing the observed climate. This study is performed over the Iberian Peninsula and focuses on the reproduction of the Probability Density Functions (PDFs) of daily mean temperature. The experiments consist on a multi-physics ensemble of high resolution climate simulations (30 km over the target region) for the periods 1970-1999 (present) and 2070-2099 (future). Two sets of simulations for the present have been performed using ERA40 (MM5-ERA40) and ECHAM5-3CM run1 (MM5-E5-PR) as boundary conditions. The future the experiments are driven by ECHAM5-A2-run1 (MM5-E5-A2). The ensemble has a total of eight members, as the result of combining the schemes for PBL (MRF and ETA), cumulus (GRELL and Kain-Fritch) and microphysics (Simple-Ice and Mixed phase). In a previous work this multi-physics ensemble has been analyzed focusing on the seasonal mean values of both temperature and precipitation. The main results indicate that those physics configurations that better reproduce the observed climate project the most dramatic changes for the future (i.e, the largest temperature increase and precipitation decrease). Among the

  6. Oscillations of cAMP with the cardiac cycle.

    PubMed

    Wikman-Coffelt, J; Sievers, R; Coffelt, R J; Parmley, W W

    1983-03-16

    Oscillations of cAMP with the cardiac cycle were demonstrated in the rat heart using a stimulator-triggered rapid freeze-clamp to decrease the temperature of the heart from 37 degrees C to -80 degrees C in 5 msec (20,000 degrees/sec) at a predetermined phase of the cardiac cycle. The nucleotide, cAMP, oscillated 60% with the cardiac cycle during normal working conditions, the higher cAMP value occurring during systole.

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

    PubMed Central

    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

  8. Mechanisms Restricting Diffusion of Intracellular cAMP.

    PubMed

    Agarwal, Shailesh R; Clancy, Colleen E; Harvey, Robert D

    2016-01-22

    Although numerous receptors stimulate cAMP production in a wide array of cells, many elicit distinct, highly localized responses, implying that the subcellular distribution of cAMP is not uniform. One often used explanation is that phosphodiesterases, which breakdown cAMP, act as functional barriers limiting diffusion. However, several studies refute the notion that this is sufficient, suggesting that phosphodiesterase-independent movement of cAMP must occur at rates slower than free diffusion. But, until now this has never been demonstrated. Using Raster Image Correlation Spectroscopy (RICS), we measured the diffusion coefficient of a fluorescently-labeled cAMP derivative (φ450-cAMP) as well as other fluorescent molecules in order to investigate the role that molecular size, cell morphology, and buffering by protein kinase A (PKA) play in restricting cAMP mobility in different cell types. Our results demonstrate that cytosolic movement of cAMP is indeed much slower than the rate of free diffusion and that interactions with PKA, especially type II PKA associated with mitochondria, play a significant role. These findings have important implications with respect to cAMP signaling in all cells.

  9. Cyclic AMP Effectors Regulate Myometrial Oxytocin Receptor Expression.

    PubMed

    Yulia, Angela; Singh, Natasha; Lei, Kaiyu; Sooranna, Suren R; Johnson, Mark R

    2016-11-01

    The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1β increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1β had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation.

  10. Revisiting cAMP signaling in the carotid body

    PubMed Central

    Nunes, Ana R.; Holmes, Andrew P.; Conde, Sílvia V.; Gauda, Estelle B.; Monteiro, Emília C.

    2014-01-01

    Chronic carotid body (CB) activation is now recognized as being essential in the development of hypertension and promoting insulin resistance; thus, it is imperative to characterize the chemotransduction mechanisms of this organ in order to modulate its activity and improve patient outcomes. For several years, and although controversial, cyclic adenosine monophosphate (cAMP) was considered an important player in initiating the activation of the CB. However, its relevance was partially displaced in the 90s by the emerging role of the mitochondria and molecules such as AMP-activated protein kinase and O2-sensitive K+ channels. Neurotransmitters/neuromodulators binding to metabotropic receptors are essential to chemotransmission in the CB, and cAMP is central to this process. cAMP also contributes to raise intracellular Ca2+ levels, and is intimately related to the cellular energetic status (AMP/ATP ratio). Furthermore, cAMP signaling is a target of multiple current pharmacological agents used in clinical practice. This review (1) provides an outline on the classical view of the cAMP-signaling pathway in the CB that originally supported its role in the O2/CO2 sensing mechanism, (2) presents recent evidence on CB cAMP neuromodulation and (3) discusses how CB activity is affected by current clinical therapies that modify cAMP-signaling, namely dopaminergic drugs, caffeine (modulation of A2A/A2B receptors) and roflumilast (PDE4 inhibitors). cAMP is key to any process that involves metabotropic receptors and the intracellular pathways involved in CB disease states are likely to involve this classical second messenger. Research examining the potential modification of cAMP levels and/or interactions with molecules associated with CB hyperactivity is currently in its beginning and this review will open doors for future explorations. PMID:25389406

  11. Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

    PubMed

    Schwede, Frank; Chepurny, Oleg G; Kaufholz, Melanie; Bertinetti, Daniela; Leech, Colin A; Cabrera, Over; Zhu, Yingmin; Mei, Fang; Cheng, Xiaodong; Manning Fox, Jocelyn E; MacDonald, Patrick E; Genieser, Hans-G; Herberg, Friedrich W; Holz, George G

    2015-07-01

    cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.

  12. Termination and activation of store-operated cyclic AMP production

    PubMed Central

    Maiellaro, Isabella; Lefkimmiatis, Konstantinos; Moyer, Mary Pat; Curci, Silvana; Hofer, Aldebaran M

    2012-01-01

    Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1-AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca2+] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store-operated’ pathway requires the ER Ca2+ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET-based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store-operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca2+ entry via the plasma membrane Ca2+ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca2+/cAMP crosstalk system. PMID:22681560

  13. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  14. Activated cAMP receptors switch encystation into sporulation.

    PubMed

    Kawabe, Yoshinori; Morio, Takahiro; James, John L; Prescott, Alan R; Tanaka, Yoshimasa; Schaap, Pauline

    2009-04-28

    Metazoan embryogenesis is controlled by a limited number of signaling modules that are used repetitively at successive developmental stages. The development of social amoebas shows similar reiterated use of cAMP-mediated signaling. In the model Dictyostelium discoideum, secreted cAMP acting on 4 cAMP receptors (cARs1-4) coordinates cell movement during aggregation and fruiting body formation, and induces the expression of aggregation and sporulation genes at consecutive developmental stages. To identify hierarchy in the multiple roles of cAMP, we investigated cAR heterogeneity and function across the social amoeba phylogeny. The gene duplications that yielded cARs 2-4 occurred late in evolution. Many species have only a cAR1 ortholog that duplicated independently in the Polysphondylids and Acytostelids. Disruption of both cAR genes of Polysphondylium pallidum (Ppal) did not affect aggregation, but caused complete collapse of fruiting body morphogenesis. The stunted structures contained disorganized stalk cells, which supported a mass of cysts instead of spores; cAMP triggered spore gene expression in Ppal, but not in the cAR null mutant, explaining its sporulation defect. Encystation is the survival strategy of solitary amoebas, and lower taxa, like Ppal, can still encyst as single cells. Recent findings showed that intracellular cAMP accumulation suffices to trigger encystation, whereas it is a complementary requirement for sporulation. Combined, the data suggest that cAMP signaling in social amoebas evolved from cAMP-mediated encystation in solitary amoebas; cAMP secretion in aggregates prompted the starving cells to form spores and not cysts, and additionally organized fruiting body morphogenesis. cAMP-mediated aggregation was the most recent innovation.

  15. Atmosphere, Magnetosphere and Plasmas in Space (AMPS). Spacelab payload definition study. Volume 3, book 2: AMPS equipment to Spacelab ICD

    NASA Technical Reports Server (NTRS)

    1976-01-01

    The interfaces between AMPS Payload No.(TBD) and Spacelab are described. The interfaces specified cover the AMPS physical, electrical, and thermal interfaces that are established to prescribe the standard Spacelab configuration required to perform the mission. If the configuration definition changes due to change of Spacelab equipment model, or serial numbers, then reidentification of the Labcraft payload may be required.

  16. Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases.

    PubMed

    de Wit, R J; Hekstra, D; Jastorff, B; Stec, W J; Baraniak, J; Van Driel, R; Van Haastert, P J

    1984-07-16

    A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS

  17. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  18. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-09

    Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

  19. Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

    SciTech Connect

    Emoto, T.; Kasai, K.; Hiraiwa, M.; Shimoda, S.

    1988-01-01

    In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E/sub 1/ or E/sub 2/ (PGE/sub 1/ and PGE/sub 2/) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10/sup -9/ M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.

  20. AMP-18 Targets p21 to Maintain Epithelial Homeostasis

    PubMed Central

    Chen, Peili; Li, Yan Chun; Toback, F. Gary

    2015-01-01

    Dysregulated homeostasis of epithelial cells resulting in disruption of mucosal barrier function is an important pathogenic mechanism in inflammatory bowel diseases (IBD). We have characterized a novel gastric protein, Antrum Mucosal Protein (AMP)-18, that has pleiotropic properties; it is mitogenic, anti-apoptotic and can stimulate formation of tight junctions. A 21-mer synthetic peptide derived from AMP-18 exhibits the same biological functions as the full-length protein and is an effective therapeutic agent in mouse models of IBD. In this study we set out to characterize therapeutic mechanisms and identify molecular targets by which AMP-18 maintains and restores disrupted epithelial homeostasis in cultured intestinal epithelial cells and a mouse model of IBD. Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine known to mediate gastrointestinal (GI) mucosal injury in IBD, was used to induce intestinal epithelial cell injury, and study the effects of AMP-18 on apoptosis and the cell cycle. An apoptosis array used to search for targets of AMP-18 in cells exposed to TNF-α identified the cyclin-dependent kinase inhibitor p21WAF1/CIP1. Treatment with AMP-18 blunted increases in p21 expression and apoptosis, while reversing disturbed cell cycle kinetics induced by TNF-α. AMP-18 appears to act through PI3K/AKT pathways to increase p21 phosphorylation, thereby reducing its nuclear accumulation to overcome the antiproliferative effects of TNF-α. In vitamin D receptor-deficient mice with TNBS-induced IBD, the observed increase in p21 expression in colonic epithelial cells was suppressed by treatment with AMP peptide. The results indicate that AMP-18 can maintain and/or restore the homeostatic balance between proliferation and apoptosis in intestinal epithelial cells to protect and repair mucosal barrier homeostasis and function, suggesting a therapeutic role in IBD. PMID:25919700

  1. Intracellular cAMP signaling by soluble adenylyl cyclase

    PubMed Central

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

    2011-01-01

    Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cAMP. sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems which are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells. PMID:21490586

  2. Cyclic AMP and the regeneration of retinal ganglion cell axons.

    PubMed

    Hellström, Mats; Harvey, Alan R

    2014-11-01

    In this paper we present a brief review of studies that have reported therapeutic benefits of elevated cAMP on plasticity and regeneration after injury to the central nervous system (CNS). We also provide new data on the cellular mechanisms by which elevation of cyclic adenosine monophosphate (cAMP) promotes cytokine driven regeneration of adult CNS axons, using the visual system as the experimental model. cAMP is a second messenger for many intracellular signalling pathways. Elevation of cAMP in the eye by intravitreal injection of the cell permeant analogue (8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate; CPT-cAMP), when added to recombinant ciliary neurotrophic factor (rCNTF), significantly enhances rCNTF-induced regeneration of adult rat retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafted onto transected optic nerve. This effect is mediated to some extent by protein kinase A (PKA) signalling, but CPT-cAMP also acts via PI3K/Akt signalling to reduce suppressor of cytokine signalling protein 3 (SOCS3) activity in RGCs. Another target for cAMP is the exchange protein activated by cAMP (Epac), which can also mediate cAMP-induced axonal growth. Here we describe some novel results and discuss to what extent the pro-regenerative effects of CPT-cAMP on adult RGCs are mediated via Epac as well as via PKA-dependent pathways. We used the established PN-optic nerve graft model and quantified the survival and regenerative growth of adult rat RGCs after intravitreal injection of rCNTF in combination with a selective activator of PKA and/or a specific activator of Epac. Viable RGCs were identified by βIII-tubulin immunohistochemistry and regenerating RGCs retrogradely labelled and quantified after an injection of fluorogold into the distal end of the PN grafts, 4 weeks post-transplantation. The specific agonists of either PKA or Epac were both effective in enhancing the effects of rCNTF on RGC axonal regeneration, but interestingly, injections

  3. Activity of cAMP-dependent protein kinases and cAMP-binding proteins of rat kidney cytosol during dehydration

    SciTech Connect

    Zelenina, M.N.; Solenov, E.I.; Ivanova, L.N.

    1985-09-20

    The activity of cAMP-dependent protein kinases, the binding of cAMP, and the spectrum of cAMP-binding proteins in the cytosol of the renal papilla was studied in intact rats and in rats after 24 h on a water-deprived diet. It was found that the activation of protein kinases by 10/sup -6/ M cAMP is significantly higher in the experimental animals than in the intact animals. In chromatography on DEAE-cellulose, the positions of the peaks of specific reception of cAMP corresponded to the peaks of the regulatory subunits of cAMP-dependent protein kinases of types I and II. In this case, in intact animals more than 80% of the binding activity was detected in peaks II, whereas in rats subjected to water deprivation, more than 60% of the binding was observed in peak I. The general regulatory activity of the cytosol was unchanged in the experimental animals in comparison with intact animals. It is suggested that during dehydration there is an induction of the synthesis of the regulatory subunit of type I cAMP-dependent protein kinase in the renal papilla.

  4. Integration of the DRAGON5/DONJON5 codes in the SALOME platform for performing multi-physics calculations in nuclear engineering

    NASA Astrophysics Data System (ADS)

    Hébert, Alain

    2014-06-01

    We are presenting the computer science techniques involved in the integration of codes DRAGON5 and DONJON5 in the SALOME platform. This integration brings new capabilities in designing multi-physics computational schemes, with the possibility to couple our reactor physics codes with thermal-hydraulics or thermo-mechanics codes from other organizations. A demonstration is presented where two code components are coupled using the YACS module of SALOME, based on the CORBA protocol. The first component is a full-core 3D steady-state neuronic calculation in a PWR performed using DONJON5. The second component implement a set of 1D thermal-hydraulics calculations, each performed over a single assembly.

  5. Atmospheric, Magnetospheric and Plasmas in Space (AMPS) spacelab payload definition study. Volume 3: Interface control documents. Part 2: AMPS payload to spacelab ICD

    NASA Technical Reports Server (NTRS)

    1976-01-01

    The AMPS to Spacelab Interface Control Document which is to be used as a guide for format and information content in generating specific AMPS Mission ICDs is presented. This document is meant to supplement the Spacelab Payload Accommodations Handbook in that it only defines interfaces which are not discussed in the handbook to the level required for design purposes. The AMPS Top Level Requirements Tree, illustrates this ICD by a shaded area and its relationship to the other AMPS technical documents. Other interface documents shown are the Level II, AMPS to Space Shuttle Vehicle ICD and the Level III, AMPS to Instruments ICD.

  6. Sustained antagonism of acute ethanol-induced ataxia following microinfusion of cyclic AMP and cpt-cAMP in the mouse cerebellum.

    PubMed

    Dar, M Saeed

    2011-05-01

    Ataxia is a conspicuous physical manifestation of alcohol consumption in humans and laboratory animals. Previously we reported possible involvement of cAMP in ethanol-induced ataxia. We now report a sustained antagonism of ataxia due to multiple ethanol injections following intracerebellar (ICB) cAMP or cpt-cAMP microinfusion. Adenylyl cyclase drugs cAMP, cpt-cAMP, Sp-cAMP, Rp-cAMP, adenosine A₁ agonist, N⁶-cyclohexyladenosine (CHA) and GABA(A) agonist muscimol were directly microinfused into the cerebellum of CD-1 male mice to evaluate their effect on ethanol (2 g/kg; i.p.) ataxia. Drug microinfusions were made via stereotaxically implanted stainless steel guide cannulas. Rotorod was used to evaluate the ethanol's ataxic response. Intracerebellar cAMP (0.1, 1, 10 fmol) or cpt-cAMP (0.5, 1, 2 fmol) 60 min before ethanol treatment, dose-dependently attenuated ethanol-induced ataxia in general agreement with previous observations. Intracerebellar microinfusion of cAMP (100 fmol) or cpt-cAMP (2 fmol) produced a sustained attenuation of ataxia following ethanol administration at 1, 4, 7 and 25 h or 31 h post-cAMP/cpt-cAMP microinfusion. At 31 h post-cAMP, the ataxic response of ethanol reappeared. Additionally, marked antagonism to the accentuation of ethanol-induced ataxia by adenosine A₁ and GABA(A) agonists, CHA (34 pmol) and muscimol (88 pmol), respectively, was noted 24h after cAMP and cpt-cAMP treatment. This indicated possible participation of AC/cAMP/PKA signaling in the co-modulation of ethanol-induced ataxia by A₁ adenosinergic and GABAergic systems. No change in normal motor coordination was noted when cAMP or cpt-cAMP microinfusion was followed by saline. Finally, Rp-cAMP (PKA inhibitor, 22 pmol) accentuated ethanol-induced ataxia and antagonized its attenuation by cAMP whereas Sp-cAMP (PKA activator, 22 pmol) produced just the opposite effects, further indicating participation of cAMP-dependent PKA downstream. Overall, the results support a role of

  7. Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.

    PubMed Central

    Fontana, D R; Luo, C S; Phillips, J C

    1991-01-01

    During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum. PMID:1846024

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

    SciTech Connect

    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.

  9. Production and release of cyclic AMP by Daphnia pulex: implications of grazing activity

    SciTech Connect

    Francko, D.A.; Wetzel, R.G.

    1982-04-01

    Daphnia pulex, a common cladoceran zooplankton species, contains tissue cAMP concentrations similar to those found in algae, bacteria, and aquatic macrophytes. Daphnia release significant quantities of cAMP into the extracellular medium. Release of algal cellular cAMP as a result of digstive degradation of algal cells may also be an important source of dissolved cAMP in lakewater.

  10. Production and release of cyclic AMP by Daphnia pulex: implications of grazing activity

    SciTech Connect

    Francko, D.A.; Wetzel, R.G.

    1982-04-01

    Daphnia pulex, a common cladoceran zooplankton species, contains tissue cAMP concentrations similar to those found in algae, bacteria, and aquatic macrophytes. Daphnia release significant quantities of cAMP into the extracellular medium. Release of algal cellular cAMP as a result of digestive degradation of algal cells may also be an important source of dissolved cAMP in lakewater.

  11. Why Ampère did not discover electromagnetic induction

    NASA Astrophysics Data System (ADS)

    Williams, L. Pearce

    1986-04-01

    In 1832, after Michael Faraday had announced his discovery of electromagnetic induction, Andre-Marie Ampère claimed that he had actually discovered the induction of one current by another in 1822. In fact, he had, but did not really publish the fact at that time. This article explores the reasons for Ampère's failure to lay claim to a discovery that would have guaranteed him scientific immortality.

  12. Microgravity changes in heart structure and cyclic-AMP metabolism

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Fine, A.; Kato, K.; Egnor, R.; Cheng, L.

    1985-01-01

    The effects of microgravity on cardiac ultrastructure and cyclic AMP metabolism in tissues of rats flown on Spacelab 3 are reported. Light and electron microscope studies of cell structure, measurements of low and high Km phosphodiesterase activity, cyclic AMP-dependent protein kinase activity, and regulatory subunit compartmentation show significant deviations in flight animals when compared to ground controls. The results indicate that some changes have occurred in cellular responses associated with catecholamine receptor interactions and intracellular signal processing.

  13. Airborne Multisensor Pod System (AMPS) data management overview

    SciTech Connect

    Wiberg, J.D.; Blough, D.K.; Daugherty, W.R.; Hucks, J.A.; Gerhardstein, L.H.; Meitzler, W.D.; Melton, R.B.; Shoemaker, S.V.

    1994-09-01

    An overview of the Data Management Plan for the Airborne Multisensor Pod System (AMPS) pro-grain is provided in this document. The Pacific Northwest Laboratory (PNL) has been assigned the responsibility of data management for the program, which includes defining procedures for data management and data quality assessment. Data management is defined as the process of planning, acquiring, organizing, qualifying and disseminating data. The AMPS program was established by the U.S. Department of Energy (DOE), Office of Arms Control and Non-Proliferation (DOE/AN) and is integrated into the overall DOE AN-10.1 technology development program. Sensors used for collecting the data were developed under the on-site inspection, effluence analysis, and standoff sensor program, the AMPS program interacts with other technology programs of DOE/NN-20. This research will be conducted by both government and private industry. AMPS is a research and development program, and it is not intended for operational deployment, although the sensors and techniques developed could be used in follow-on operational systems. For a complete description of the AMPS program, see {open_quotes}Airborne Multisensor Pod System (AMPS) Program Plan{close_quotes}. The primary purpose of the AMPS is to collect high-quality multisensor data to be used in data fusion research to reduce interpretation problems associated with data overload and to derive better information than can be derived from any single sensor. To collect the data for the program, three wing-mounted pods containing instruments with sensors for collecting data will be flight certified on a U.S. Navy RP-3A aircraft. Secondary objectives of the AMPS program are sensor development and technology demonstration. Pod system integrators and instrument developers will be interested in the performance of their deployed sensors and their supporting data acquisition equipment.

  14. Allostery and Conformational Dynamics in cAMP-binding Acyltransferases*

    PubMed Central

    Podobnik, Marjetka; Siddiqui, Nida; Rebolj, Katja; Nambi, Subhalaxmi; Merzel, Franci; Visweswariah, Sandhya S.

    2014-01-01

    Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein. PMID:24748621

  15. Allostery and conformational dynamics in cAMP-binding acyltransferases.

    PubMed

    Podobnik, Marjetka; Siddiqui, Nida; Rebolj, Katja; Nambi, Subhalaxmi; Merzel, Franci; Visweswariah, Sandhya S

    2014-06-06

    Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.

  16. Chemotactic responses of Dictyostelium discoideum amoebae to a cyclic AMP concentration gradient: evidence to support a spatial mechanism for sensing cyclic AMP.

    PubMed

    Tani, T; Naitoh, Y

    1999-01-01

    The motile responses of Dictyostelium discoideum amoebae to a cyclic AMP (cAMP) concentration gradient were examined using a novel assay system. In this system, a cAMP concentration gradient was generated, while the overall cAMP concentration could be either increased or decreased in a chamber containing amoebae. The chemotactic responses of amoebae were examined immediately after they had been subjected to the cAMP concentration gradient. Amoebae moving in random directions in a reference solution ascended a cAMP concentration gradient after they had been exposed to the gradient irrespective of whether there was an increase or a decrease in the overall cAMP concentration. This strongly supports the idea that D. discoideum amoebae can sense a spatial cAMP gradient around them and that this causes their chemoaccumulation behavior. Ascending locomotion became less conspicuous when the amoebae were treated with a homogeneous cAMP solution for approximately 8 min before exposure to a cAMP gradient. This cAMP pretreatment reduced the sensitivity of the amoeba to a cAMP concentration gradient. The cAMP concentration gradient could be reversed in less than 30 s in this assay system, allowing the generation of a cAMP wave by accumulating amoebae to be mimicked. The ascending amoebae continued to move in the same direction for 1-2 min after the gradient had been reversed. This is consistent with the well-known observation that reversal of a cAMP concentration gradient experienced by the amoebae passing through a cAMP wave does not negate their chemotactic movement towards the accumulation center.

  17. Cyclic AMP and cell division in Escherichia coli.

    PubMed Central

    D'Ari, R; Jaffé, A; Bouloc, P; Robin, A

    1988-01-01

    We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex. Images PMID:2826407

  18. Intracellular tortuosity underlies slow cAMP diffusion in adult ventricular myocytes

    PubMed Central

    Richards, Mark; Lomas, Oliver; Jalink, Kees; Ford, Kerrie L.; Vaughan-Jones, Richard D.; Lefkimmiatis, Konstantinos; Swietach, Pawel

    2016-01-01

    Aims 3′,5′-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (DcAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. Methods and results [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of DcAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, DcAMP was calculated to be 32 µm2/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate DcAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. Conclusion In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling. PMID:27089919

  19. Neuronal activity promotes myelination via a cAMP pathway.

    PubMed

    Malone, Misti; Gary, Devin; Yang, In Hong; Miglioretti, Anna; Houdayer, Thierry; Thakor, Nitish; McDonald, John

    2013-06-01

    Neuronal activity promotes myelination in vivo and in vitro. However, the molecular events that mediate activity-dependent myelination are not completely understood. Seven, daily 1 h sessions of patterned electrical stimulation (ESTIM) promoted myelin segment formation in mixed cultures of dorsal root ganglion (DRG) neurons and oligodendrocytes (OLs); the increase in myelination was frequency-dependent. Myelin segment formation was also enhanced following exposure of DRGs to ESTIM prior to OL addition, suggesting that ESTIM promotes myelination in a manner involving neuron-specific signaling. Cyclic adenosine monophosphate (cAMP) levels in DRGs were increased three-fold following ESTIM, and artificially increasing cAMP mimicked the ability of ESTIM to promote myelination. Alternatively, inhibiting the cAMP pathway suppressed ESTIM-induced myelination. We used compartmentalized, microfluidic platforms to isolate DRG soma from OLs and assessed cell-type specific effects of ESTIM on myelination. A selective increase or decrease in DRG cAMP levels resulted in enhanced or suppressed myelination, respectively. This work describes a novel role for the cAMP pathway in neurons that results in enhanced myelination.

  20. AKAP18 contains a phosphoesterase domain which binds AMP

    PubMed Central

    Gold, Matthew G.; Smith, F. Donelson; Scott, John D.; Barford, David

    2011-01-01

    SUMMARY Protein kinase A anchoring proteins (AKAPs), defined by their capacity to target the cAMP-dependent protein kinase to distinct sub-cellular locations, function as molecular scaffolds mediating the assembly of multi-component complexes to integrate and organise multiple signalling events. Despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. Here, using bioinformatics and X-ray crystallography, we define a central domain of AKAP18δ (AKAP18CD) as a member of the 2H phosphoesterase family. The domain features two conserved His-x-Thr motifs positioned at the base of a groove located between two lobes related by pseudo two-fold symmetry. Nucleotide co-crystallisation screening revealed that this groove binds specifically to 5’AMP/CMP, with the affinity constant for AMP in the physiological concentration range. This is the first example of an AKAP capable of binding a small molecule. Our data generate two functional hypotheses for the AKAP18 central domain. It may act as a phosphoesterase, although we did not identify a substrate, or as an AMP sensor with the potential to couple intracellular AMP levels to PKA signalling events. PMID:18082768

  1. Altered AMP deaminase activity may extend postmortem glycolysis.

    PubMed

    England, E M; Matarneh, S K; Scheffler, T L; Wachet, C; Gerrard, D E

    2015-04-01

    Postmortem energy metabolism drives hydrogen accumulation in muscle and results in a fairly constant ultimate pH. Extended glycolysis results in adverse pork quality and may be possible with greater adenonucleotide availability postmortem. We hypothesized that slowing adenonucleotide removal by reducing AMP deaminase activity would extend glycolysis and lower the ultimate pH of muscle. Longissimus muscle samples were incorporated into an in vitro system that mimics postmortem glycolysis with or without pentostatin, an AMP deaminase inhibitor. Pentostatin lowered ultimate pH and increased lactate and glucose 6-phosphate with time. Based on these results and that AMPK γ3(R200Q) mutated pigs (RN⁻) produce low ultimate pH pork, we hypothesized AMP deaminase abundance and activity would be lower in RN⁻ muscle than wild-type. RN⁻ muscle contained lower AMP deaminase abundance and activity. These data show that altering adenonucleotide availability postmortem can extend postmortem pH decline and suggest that AMP deaminase activity may, in part, contribute to the low ultimate pH observed in RN⁻ pork.

  2. The regulation of AMP-activated protein kinase by phosphorylation.

    PubMed Central

    Stein, S C; Woods, A; Jones, N A; Davison, M D; Carling, D

    2000-01-01

    The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (alpha) of AMPK (Thr(172)) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr(172) on AMPK activity. Mutation of Thr(172) to an aspartic acid residue (T172D) in either alpha1 or alpha2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr(172) to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr(172) accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the alpha subunit and one site on the beta subunit. Furthermore, we provide evidence that phosphorylation of Thr(172) may be involved in the sensitivity of the AMPK complex to AMP. PMID:10642499

  3. Cyclic nucleotide phosphodiesterase-mediated integration of cGMP and cAMP signaling in cells of the cardiovascular system.

    PubMed

    Maurice, Donald H

    2005-05-01

    Numerous pharmacological and physiological agents acting via either cAMP- or cGMP-mediated impact the activities of cells of the cardiovascular system. While most define cAMP and cGMP signaling systems as separate and independent, recent advances in our understanding of cyclic nucleotide signaling, and more specifically, of the roles which cyclic nucleotide phosphodiesterases (PDEs) play in these events, have altered this view. In this short chapter, I will review the data identifying expression of several PDEs in cells of the cardiovascular system. In addition, I will review the data that identify PDEs as enzymes capable of allowing integration between cAMP and cGMP signaling in cells, and propose that cAMP and cGMP signaling systems can represent parallel and interdependent signaling systems. Moreover, I will propose that cGMP-mediated effects on the activities of variants of the Phosphodiesterase 2 (PDE2), PDE3 and PDE5 families may act to coordinate linkage between cAMP and cGMP signaling in these cells.

  4. 1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP as probes of the activator site of glycogen phosphorylase from rabbit skeletal muscle.

    PubMed Central

    Vandenbunder, B; Morange, M; Buc, H

    1976-01-01

    Both 1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP bind at the AMP site of phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Etheno-AMP induces the same activation as AMP, about 30-fold higher than the activation induced by etheno-dAMP. The fluorescence of etheno-AMP and etheno-dAMP is associated with the base moiety; therefore, when free in solution, the two derivatives have identical fluorescence properties. However, when bound to phosphorylase, the fluorescence of etheno-AMP is quenched more efficiently than the fluorescence of etheno-dAMP. This difference between the fluorescence properties of the bound nucleotides suggests that a modification in the ribose ring affects the position of the adenine in the AMP site of phosphorylase b. The observed quenching may be due to a stacking interaction between an aromatic residue and the base moiety of the bound nucleotide. PMID:1066682

  5. Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system.

    PubMed

    Stangherlin, Alessandra; Zaccolo, Manuela

    2012-01-01

    Phosphodiesterases are key enzymes in the cAMP signaling cascade. They convert cAMP in its inactive form 5'-AMP and critically regulate the intensity and the duration of cAMP-mediated signals. Multiple isoforms exist that possess different intracellular distributions, different affinities for cAMP, and different catalytic and regulatory properties. This complex repertoire of enzymes provides a multiplicity of ways to modulate cAMP levels, to integrate more signaling pathways, and to respond to the specific needs of the cell within distinct subcellular domains. In this review we summarize key findings on phosphodiesterase compartmentalization in the cardiovascular system.

  6. Intracellular cAMP signaling by soluble adenylyl cyclase.

    PubMed

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

    2011-06-01

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

  7. Cyclic AMP Signaling: A Molecular Determinant of Peripheral Nerve Regeneration

    PubMed Central

    Knott, Eric P.; Assi, Mazen; Pearse, Damien D.

    2014-01-01

    Disruption of axonal integrity during injury to the peripheral nerve system (PNS) sets into motion a cascade of responses that includes inflammation, Schwann cell mobilization, and the degeneration of the nerve fibers distal to the injury site. Yet, the injured PNS differentiates itself from the injured central nervous system (CNS) in its remarkable capacity for self-recovery, which, depending upon the length and type of nerve injury, involves a series of molecular events in both the injured neuron and associated Schwann cells that leads to axon regeneration, remyelination repair, and functional restitution. Herein we discuss the essential function of the second messenger, cyclic adenosine monophosphate (cyclic AMP), in the PNS repair process, highlighting the important role the conditioning lesion paradigm has played in understanding the mechanism(s) by which cyclic AMP exerts its proregenerative action. Furthermore, we review the studies that have therapeutically targeted cyclic AMP to enhance endogenous nerve repair. PMID:25177696

  8. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    NASA Technical Reports Server (NTRS)

    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.

  9. Inhibition of AMP deaminase as therapeutic target in cardiovascular pathology.

    PubMed

    Zabielska, Magdalena A; Borkowski, Tomasz; Slominska, Ewa M; Smolenski, Ryszard T

    2015-08-01

    AMP deaminase (AMPD; EC 3.5.4.6) catalyzes hydrolysis of the amino group from the adenine ring of AMP resulting in production of inosine 5'-monophosphate (IMP) and ammonia. This reaction helps to maintain healthy cellular energetics by removing excess AMP that accumulates in energy depleted cells. Furthermore, AMPD permits the synthesis of guanine nucleotides from the larger adenylate pool. This enzyme competes with cytosolic 5'-nucleotidases (c5NT) for AMP. Adenosine, a product of c5NT is a vasodilator, antagonizes inotropic effects of catecholamines and exerts anti-platelet, anti-inflammatory and immunosuppressive activities. The ratio of AMPD/c5NT defines the amount of adenosine produced in adenine nucleotide catabolic pathway. Inhibition of AMPD could alter this ratio resulting in increased adenosine production. Besides the potential effect on adenosine production, elevation of AMP due to inhibition of AMPD could also lead to activation of AMP regulated protein kinase (AMPK) with myriad of downstream events including enhanced energetic metabolism, mitochondrial biogenesis and cytoprotection. While the benefits of these processes are well appreciated in cells such as skeletal or cardiac myocytes its role in protection of endothelium could be even more important. Therapeutic use of AMPD inhibition has been limited due to difficulties with obtaining compounds with adequate characteristics. However, endothelium seems to be the easiest target as effective inhibition of AMPD could be achieved at much lower concentration than in the other types of cells. New generation of AMPD inhibitors has recently been established and its testing in context of endothelial and organ protection could provide important basic knowledge and potential therapeutic tools.

  10. Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.

    PubMed

    Khan, D R; Guillemette, C; Sirard, M A; Richard, F J

    2015-09-01

    Acquisition of oocyte developmental competence needs to be understood to improve clinical outcomes of assisted reproduction. The stimulation of cumulus cell concentration of cyclic adenosine 3'5'-monophosphate (cAMP) by pharmacological agents during in vitro maturation (IVM) participates in improvement of oocyte quality. However, precise coordination and downstream targets of cAMP signaling in cumulus cells are largely unknown. We have previously demonstrated better embryo development after cAMP stimulation for first 6 h during IVM. Using this model, we investigated cAMP signaling in cumulus cells through in vitro culture of cumulus-oocyte complexes (COCs) in the presence of cAMP raising agents: forskolin, IBMX, and dipyridamole (here called FID treatment). Transcriptomic analysis of cumulus cells indicated that FID-induced differentially expressed transcripts were implicated in cumulus expansion, steroidogenesis, cell metabolism, and oocyte competence. Functional genomic analysis revealed that protein kinase-A (PKA), extracellular signal regulated kinases (ERK1/2), and calcium (Ca(2+)) pathways as key regulators of FID signaling. Inhibition of PKA (H89) in FID-supplemented COCs or substitution of FID with calcium ionophore (A23187) demonstrated that FID activated primarily the PKA pathway which inhibited ERK1/2 phosphorylation and was upstream of calcium signaling. Furthermore, inhibition of ERK1/2 phosphorylation by FID supported a regulation by dual specific phosphatase (DUSP1) via PKA. Our findings imply that cAMP (FID) regulates cell metabolism, steroidogenesis, intracellular signaling and cumulus expansion through PKA which modulates these functions through optimization of ERK1/2 phosphorylation and coordination of calcium signaling. These findings have implications for development of new strategies for improving oocyte in vitro maturation leading to better developmental competence.

  11. Regulation and organization of adenylyl cyclases and cAMP.

    PubMed Central

    Cooper, Dermot M F

    2003-01-01

    Adenylyl cyclases are a critically important family of multiply regulated signalling molecules. Their susceptibility to many modes of regulation allows them to integrate the activities of a variety of signalling pathways. However, this property brings with it the problem of imparting specificity and discrimination. Recent studies are revealing the range of strategies utilized by the cyclases to solve this problem. Microdomains are a consequence of these solutions, in which cAMP dynamics may differ from the broad cytosol. Currently evolving methodologies are beginning to reveal cAMP fluctuations in these various compartments. PMID:12940771

  12. AKAPs: The Architectural Underpinnings of Local cAMP signaling

    PubMed Central

    Kritzer, Michael D.; Li, Jinliang; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

    2011-01-01

    The cAMP-dependent protein kinase A (PKA) is targeted to specific compartments in the cardiac myocyte by A-kinase anchoring proteins (AKAPs), a diverse set of scaffold proteins that have been implicated in the regulation of excitation-contraction coupling and cardiac remodeling. AKAPs bind not only PKA, but also a large variety of structural and signaling molecules. In this review, we discuss the basic concepts underlying compartmentation of cAMP and PKA signaling, as well as a few of the individual AKAPs that have been shown to be functionally relevant in the heart. PMID:21600214

  13. “cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

    PubMed Central

    Lefkimmiatis, Konstantinos; Moyer, Mary Pat; Curci, Silvana; Hofer, Aldebaran M.

    2009-01-01

    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 RIβ 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. PMID:19888343

  14. Plasmid-Encoded AmpC (pAmpC) in Enterobacteriaceae: epidemiology of microorganisms and resistance markers.

    PubMed

    Cejas, Daniela; Fernández Canigia, Liliana; Quinteros, Mirta; Giovanakis, Marta; Vay, Carlos; Lascialandare, Silvana; Mutti, Daniel; Pagniez, Gastón; Almuzara, Marisa; Gutkind, Gabriel; Radice, Marcela

    2012-01-01

    CMY-2 Β-lactamase is an important cause of Β-lactam resistance in Enterobacteriaceae and constitutes the most widespread pAmpC. Although CMY-2 has been previously recognized in our region, the real prevalence and epidemiology of this resistance marker was uncertain. During August-October 2009, we conducted a multicenter, prospective study to determine pAmpC prevalence and to characterize CMY-2 producing Escherichia coli associated plasmids. Plasmid-encoded AmpC prevalence was 0.9 % in enterobacteria in this period, being CMY-2 prevalent and to a lesser extent DHA. Molecular typing of CMY-2- producing Escherichia coli isolates showed several lineages. Moreover, replicon typing of cmy-2- containing plasmids displayed a broad diversity in Inc/cmy-2 links. Therefore, association of cmy-2 with specific transposon elements may be responsible for the spread of this resistance marker in Enterobacteriaceae.

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

    PubMed Central

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

    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 fusions. The reduced expression of tfoXVC was restored by trans-complementation of crp in ∆crp and by exogenous cAMP in ∆cya. A promoter deletion analysis and the site-directed mutagenesis of the putative CRP-N sites revealed the presence of two functional CRP-N sites. The direct binding of cAMP-CRP to the tfoXVCpromoter was demonstrated by EMSA assays. Additionally, the transcriptional start site (TSS) of tfoXVF in V. fluvialis was determined, and −10/−35 regions were predicted. Further comparison of the tfoX promoter in Vibrios revealed the existence of similar −10 motifs and putative CRP-N sites, indicating the conserved mechanism of CRP regulation on tfoX. Our study demonstrates the direct binding of the cAMP-CRP complex to tfoX promoter, and broadens the understanding of the molecular mechanism regulating tfoX in Vibrios. PMID:26442598

  16. Field measurements and interpretation of TMI-2 instrumentation: YM-AMP-7023 and YM-AMP-7025

    SciTech Connect

    Jones, J E; Smith, J T; Mathis, M V

    1982-01-01

    This report describes the measurement and results of the Loose Part Monitor Channels YM-AMP-7023 and YM-AMP-7025. These instruments consist of an Endevco Model 2276 accelerometer and a model 2652M4 charge amplifier connected to the Loose Parts Monitorng System terminals by approximately 400 feet (500 feet for 7025) of cable. The instruments were being incorporated into a B and W supplied system when the measurements were taken; therefore, the equipment was not expected to be fully operational.

  17. The cyclic AMP (cAMP)-cAMP receptor protein signaling system mediates resistance of Vibrio cholerae O1 strains to multiple environmental bacteriophages.

    PubMed

    Zahid, M Shamim Hasan; Waise, T M Zaved; Kamruzzaman, M; Ghosh, Amar N; Nair, G Balakrish; Mekalanos, John J; Faruque, Shah M

    2010-07-01

    Toxigenic Vibrio cholerae, the causative agent of the epidemic diarrheal disease cholera, interacts with diverse environmental bacteriophages. These interactions promote genetic diversity or cause selective enrichment of phage-resistant bacterial clones. To identify bacterial genes involved in mediating the phage-resistant phenotype, we screened a transposon insertion library of V. cholerae O1 El Tor biotype strain C6706 to identify mutants showing altered susceptibility to a panel of phages isolated from surface waters in Bangladesh. Mutants with insertion in cyaA or crp genes encoding adenylate cyclase or cyclic AMP (cAMP) receptor protein (CRP), respectively, were susceptible to a phage designated JSF9 to which the parent strain was completely resistant. Application of the cyaA mutant as an indicator strain in environmental phage monitoring enhanced phage detection, and we identified 3 additional phages to which the parent strain was resistant. Incorporation of the cyaA or crp mutations into other V. cholerae O1 strains caused similar alterations in their phage susceptibility patterns, and the susceptibility correlated with the ability of the bacteria to adsorb these phages. Our results suggest that cAMP-CRP-mediated downregulation of phage adsorption may contribute to a mechanism for the V. cholerae O1 strains to survive predation by multiple environmental phages. Furthermore, the cyaA or crp mutant strains may be used as suitable indicators in monitoring cholera phages in the water.

  18. Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.).

    PubMed

    Fujimura, Masatoshi; Ideguchi, Mineo; Minami, Yuji; Watanabe, Keiichi; Tadera, Kenjiro

    2004-03-01

    Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.

  19. Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

    PubMed Central

    Walter, U; Costa, M R; Breakefield, X O; Greengard, P

    1979-01-01

    Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells. Images PMID:226964

  20. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  1. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  2. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  3. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  4. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  5. Spatial encoding of cyclic AMP signalling specificity by GPCR endocytosis

    PubMed Central

    Tsvetanova, Nikoleta G.; von Zastrow, Mark

    2014-01-01

    G protein-coupled receptors (GPCRs) are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various GPCRs also signal after internalization. Apart from its temporal impact through prolonging the cellular response, does the endosome-initiated signal encode any discrete spatial information? Using the beta2-adrenoceptor (β2-AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP-dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Finally, our results suggest that this spatial encoding scheme helps cells functionally discriminate chemically distinct β2-AR ligands according to differences in their ability to promote receptor endocytosis. These findings reveal a discrete principle for achieving cellular signalling specificity, based on endosome-mediated spatial encoding of intracellular second messenger production and ‘location aware’ downstream transcriptional control. PMID:25362359

  6. Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

    PubMed

    Fontana, D R; Price, P L; Phillips, J C

    1991-01-01

    Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Multi-initial-conditions and Multi-physics Ensembles in the Weather Research and Forecasting Model to Improve Coastal Stratocumulus Forecasts for Solar Power Integration

    NASA Astrophysics Data System (ADS)

    Yang, H.

    2015-12-01

    used to create a multi-parameter and multi-physics ensemble. The ensemble forecast system is implemented operationally for San Diego Gas & Electric Company to improve system operations.

  8. Structure-Function Analysis of the Transmembrane Protein AmpG from Pseudomonas aeruginosa

    PubMed Central

    Li, Peizhen; Ying, Jun; Yang, Guangjian; Li, Aifang; Wang, Jian; Lu, Junwan; Wang, Junrong; Xu, Teng; Yi, Huiguang; Li, Kewei; Jin, Shouguang; Bao, Qiyu; Zhang, Kaibo

    2016-01-01

    AmpG is a transmembrane protein with permease activity that transports meuropeptide from the periplasm to the cytoplasm, which is essential for the induction of the ampC encoding β-lactamase. To obtain new insights into the relationship between AmpG structure and function, comparative genomics analysis, secondary and tertiary structure modeling, site-directed mutational analyses and genetic complementation experiments were performed in this study. AmpGs from different genera of bacteria (Escherichia coli, Vibrio cholerae and Acinetobacter baumannii) could complement AmpG function in Pseudomonas aeruginosa. The minimal inhibitory concentration (MIC) to ampicillin is 512 μg/ml for wild type strain PAO1, while it is 32 μg/ml for an ampG deletion mutant strain (PAO1ΔampG) with a corresponding decrease in the activity of the ampC-encoded β-lactamase. Site-directed mutagenesis of conserved AmpG residues (G29, A129, Q131 and A197) resulted in a loss of function, resulting in a loss of resistance to ampicillin in PAO1ΔampG. The G29A, G29V, A129T, A129V, A129D, A197S and A197D mutants had lower resistance to ampicillin and significantly decreased activity of the AmpC β-lactamase. The G29A, G29V, A129V, A197S and A197D mutants had decreased ampG mRNA transcript levels. The A129T and A129D mutants had normal ampG mRNA transcript levels, but the function of the protein was drastically reduced. Our experimental results demonstrate that the conserved amino acids played essential roles in maintaining the function of AmpG. Combined with the AmpG structural information, these critical amino acids can be targeted for the development of new anti-bacterial agents. PMID:27959942

  9. Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

    PubMed

    Kuno, T; Shuntoh, H; Sakaue, M; Saijoh, K; Takeda, T; Fukuda, K; Tanaka, C

    1988-06-30

    The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

  10. Cyclic AMP and its functional relationship in Tetrahymena: a comparison between phagocytosis and glucose uptake.

    PubMed

    Csaba, G; Nagy, S U; Lantos, T

    1978-01-01

    In Tetrahymena, an increase in the level of cAMP is accompanied by an increased phagocytotic rate, whereas increased sugar uptake is parallelled by a decreased cAMP level. The increase in cAMP level seems to be decisive with respect to phagocytosis as a basic phenomenon of life. In the action of epinephrine, however, some mechanism other than cAMP mediation may be involved. Depending on concentration, one hormone may provoke either an increase or a decrease in cAMP level, and this in turn triggers the corresponding function.

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

    PubMed Central

    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. PMID:26361388

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

    PubMed

    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.

  13. Pre-amp EDFA noise characterization for optimal optical receiver transmission performance

    NASA Astrophysics Data System (ADS)

    Abu-Aisheh, Akram Ahmad

    In fiber optic communication systems, a pre-amp Erbium Doped Fiber Amplifier (EDFA) is used at the input of the optical receiver to increase the receiver sensitivity by amplifying the photon detector incoming optical signal. As a result of this amplification process, the performance of the photon detector is degraded by the pre-amp noise. So, it is important to characterize the pre-amp noise at the optical receiver level and relate the pre-amp noise performance to the optical receiver transmission performance. In this dissertation, the pre-amp EDFA noise performance was characterized first at the pre-amp level through modeling using computer simulations. Then, the pre-amp noise performance was characterized experimentally at the optical receiver level. This dissertation demonstrates that simulations and experiments together provide the optimization of the pre-amp EDFA performance. The experimental work of this dissertation focused on the pre-amp EDFA noise performance characterization and analysis at the optical receiver level. This is the ultimate performance characterization method for the pre-amp EDFA, and it was performed through testing the optical receiver transmission performance under different pre-amp operating conditions.

  14. cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity.

    PubMed

    Choi, H S; Li, B; Lin, Z; Huang, E; Liu, A Y

    1991-06-25

    The theme of this study is an evaluation of the involvement of cAMP and cAMP-dependent protein kinase (PKA) in the regulation of the human heat shock protein (hsp) 70 gene promoter. Expression of a highly specific protein inhibitor of PKA (pRSVPKI) inhibited the basal as well as heat- and cadmium-induced expression of the cotransfected pHBCAT, a human hsp 70 promoter-driven reporter gene; this inhibition was dependent on the amount of pRSVPKI used. The effect of an expression vector of the RI regulatory subunit of PKA, pMTREV, was similar to that of pRSVPKI; pMTREV inhibited both the basal as well as the heat-induced expression of pHBCAT. The specificity of effects of these expression vectors was demonstrated by the lack of effect of a mutant PKI gene and by the unaffected expression of a reference gene (pRSV beta gal) under these conditions. Analysis of the effects of dibutyryl cAMP (1 mM), forskolin (10 microM), and 8-Br-cAMP (1 mM) on the transient expression of pHBCAT showed that these cAMP-elevating agents stimulated the hsp 70 promoter activity, whereas cAMP (1 mM) was without effect. Chloramphenicol acetyltransferase gene constructs with truncated or mutated hsp 70 promoter were used to define the cis-acting DNA element(s) that confer this cAMP stimulation; the heat induced (42 degrees C) expression was used as a control. Mutation of the adenovirus transcription factor element (pLSN-40/-26) greatly reduced the basal level of expression; forskolin had little or no effect on this adenovirus transcription factor-minus promoter, although the promoter activity was very heat inducible. The absence of a functional heat shock consensus element (HSE) in the construct pLSPNWT rendered the promoter heat insensitive; this construct was forskolin responsive although the magnitude of this stimulation was reduced when compared with that of a control construct with HSE. These results were corroborated by studies using consensus sequence of ATF (ATFE) and HSE as competitors

  15. Altering cAMP levels within a central pattern generator modifies or disrupts rhythmic motor output.

    PubMed

    Clemens, Stefan; Calin-Jageman, Robert; Sakurai, Akira; Katz, Paul S

    2007-12-01

    Cyclic AMP is a second messenger that has been implicated in the neuromodulation of rhythmically active motor patterns. Here, we tested whether manipulating cAMP affects swim motor pattern generation in the mollusc, Tritonia diomedea. Inhibiting adenylyl cyclase (AC) with 9-cyclopentyladenine (9-CPA) slowed or stopped the swim motor pattern. Inhibiting phosphodiesterase with 3-isobutyl-1-methylxanthine (IBMX) or applying dibutyryl-cAMP (dB-cAMP) disrupted the swim motor pattern, as did iontophoresing cAMP into the central pattern generator neuron C2. Additionally, during wash-in, IBMX sometimes temporarily produced extended or spontaneous swim motor patterns. Photolysis of caged cAMP in C2 after initiation of the swim motor pattern inhibited subsequent bursting. These results suggest that cAMP levels can dynamically modulate swim motor pattern generation, possibly shaping the output of the central pattern generator on a cycle-by-cycle basis.

  16. Aerobic Methane Generation From Plants (AMP)? Yes, Mostly!

    NASA Astrophysics Data System (ADS)

    Whiticar, M. J.; Ednie, A. C.

    2007-12-01

    In 2006, Keppler et al. (K) published an intriguing and revolutionary idea that aerobic methane is produced in plants (AMP) and released to the atmosphere. Their initial scaling calculations estimated the amount of AMP fluxing from living plants to range from 62-236 Tg/y and 1-7 Tg/y for plant litter. Houweling et al. (2006) (H) refined this flux to ca. 85 Tg/y PIH and 125 Tg/y present day. More recently, Dueck et al. (2007) (D) challenged the claim of AMP from intact plants. Their experiments cited "...No evidence for substantial aerobic methane emission by terrestrial plants..." (max. 0.4 ng/g h-1). Due to the significance of AMP in understanding present and palaeo-atmospheric budgets (e.g., Whiticar and Schaefer, 2007), we conducted a wide range of experiments to confirm or refute the existence and magnitude of AMP. For explanation, experiments of K were time-series batch samples measured by gas chromatography on purged and ambient samples, whereas D used continuous-flow cuvettes and measured by optical PAS with time series single injections. Our longer-term experiments with corn, wheat, tomato, red cedar, chestnut, moss and lichen (3-97 h, 32 °C) used a plant chamber, flow-through system with a GYRO, an optical spectrometer that enables continuous 1 Hz CH4 measurements with a precision of ca. 1 ppbv. We conducted over 100 chamber experiments on sterilized and non-sterilized (Cs-137 radiation) samples of: 1) intact living plants (IP), 2) fresh leaves (FL) and 3) dried leaves (DL); under both 1) high and 2) low light conditions (HL, LL), and with 1) ambient CH4 (AM, ca. 1.92 ppmv) and 2) purged methane (PM, 10 and 96 ppbv) levels. Our results demonstrate that IP-AMs have CH4 flux rates of 0.74-3.48 ng/g h-1. In contrast, IP-PMs show intense CH4 uptake rates of -28.5 to -57.9 ng/g h-1 (substantially different than K's reported emissions of 12-370 ng/g h-1 values). Our FL-AM-LL have CH4 flux rates of 0.36-2.05 ng/g h-1, whereas FL-AM-HL have significant CH4

  17. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Advances in Simulation of Wave Interaction with Extended MHD Phenomena

    SciTech Connect

    Batchelor, Donald B; Abla, Gheni; D'Azevedo, Ed F; Bateman, Glenn; Bernholdt, David E; Berry, Lee A; Bonoli, P.; Bramley, R; Breslau, Joshua; Chance, M.; Chen, J.; Choi, M.; Elwasif, Wael R; Foley, S.; Fu, GuoYong; Harvey, R. W.; Jaeger, Erwin Frederick; Jardin, S. C.; Jenkins, T; Keyes, David E; Klasky, Scott A; Kruger, Scott; Ku, Long-Poe; Lynch, Vickie E; McCune, Douglas; Ramos, J.; Schissel, D.; Schnack,; Wright, J.

    2009-01-01

    The Integrated Plasma Simulator (IPS) provides a framework within which some of the most advanced, massively-parallel fusion modeling codes can be interoperated to provide a detailed picture of the multi-physics processes involved in fusion experiments. The presentation will cover four topics: 1) recent improvements to the IPS, 2) application of the IPS for very high resolution simulations of ITER scenarios, 3) studies of resistive and ideal MHD stability in tokamk discharges using IPS facilities, and 4) the application of RF power in the electron cyclotron range of frequencies to control slowly growing MHD modes in tokamaks and initial evaluations of optimized location for RF power deposition.

  20. Advances in Simulation of Wave Interactions with Extended MHD Phenomena

    SciTech Connect

    Batchelor, Donald B; D'Azevedo, Eduardo; Bateman, Glenn; Bernholdt, David E; Bonoli, P.; Bramley, Randall B; Breslau, Joshua; Elwasif, Wael R; Foley, S.; Jaeger, Erwin Frederick; Jardin, S. C.; Klasky, Scott A; Kruger, Scott E; Ku, Long-Poe; McCune, Douglas; Ramos, J.; Schissel, David P; Schnack, Dalton D

    2009-01-01

    The Integrated Plasma Simulator (IPS) provides a framework within which some of the most advanced, massively-parallel fusion modeling codes can be interoperated to provide a detailed picture of the multi-physics processes involved in fusion experiments. The presentation will cover four topics: (1) recent improvements to the IPS, (2) application of the IPS for very high resolution simulations of ITER scenarios, (3) studies of resistive and ideal MHD stability in tokamak discharges using IPS facilities, and (4) the application of RF power in the electron cyclotron range of frequencies to control slowly growing MHD modes in tokamaks and initial evaluations of optimized location for RF power deposition.

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

    PubMed

    Lanaspa, Miguel A; Epperson, L Elaine; Li, Nanxing; Cicerchi, Christina; Garcia, Gabriela E; Roncal-Jimenez, Carlos A; Trostel, Jessica; Jain, Swati; Mant, Colin T; Rivard, Christopher J; Ishimoto, Takuji; Shimada, Michiko; Sanchez-Lozada, Laura Gabriela; Nakagawa, Takahiko; Jani, Alkesh; Stenvinkel, Peter; Martin, Sandra L; Johnson, Richard J

    2015-01-01

    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.

  2. Exchange protein activated by cAMP (Epac) mediates cAMP-dependent but protein kinase A-insensitive modulation of vascular ATP-sensitive potassium channels.

    PubMed

    Purves, Gregor I; Kamishima, Tomoko; Davies, Lowri M; Quayle, John M; Dart, Caroline

    2009-07-15

    Exchange proteins directly activated by cyclic AMP (Epacs or cAMP-GEF) represent a family of novel cAMP-binding effector proteins. The identification of Epacs and the recent development of pharmacological tools that discriminate between cAMP-mediated pathways have revealed previously unrecognized roles for cAMP that are independent of its traditional target cAMP-dependent protein kinase (PKA). Here we show that Epac exists in a complex with vascular ATP-sensitive potassium (KATP) channel subunits and that cAMP-mediated activation of Epac modulates KATP channel activity via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive protein phosphatase 2B (PP-2B, calcineurin). Application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP, at concentrations that activate Epac but not PKA, caused a 41.6 +/- 4.7% inhibition (mean +/- S.E.M.; n = 7) of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Importantly, similar results were obtained when cAMP was elevated by addition of the adenylyl cyclase activator forskolin in the presence of the structurally distinct PKA inhibitors, Rp-cAMPS or KT5720. Activation of Epac by 8-pCPT-2'-O-Me-cAMP caused a transient 171.0 +/- 18.0 nM (n = 5) increase in intracellular Ca2+ in Fura-2-loaded aortic myocytes, which persisted in the absence of extracellular Ca2+. Inclusion of the Ca2+-specific chelator BAPTA in the pipette-filling solution or preincubation with the calcineurin inhibitors, cyclosporin A or ascomycin, significantly reduced the ability of 8-pCPT-2'-O-Me-cAMP to inhibit whole-cell KATP currents. These results highlight a previously undescribed cAMP-dependent regulatory mechanism that may be essential for understanding the physiological and pathophysiological roles ascribed to arterial KATP channels in the control of vascular tone and blood flow.

  3. A novel biosensor to study cAMP dynamics in cilia and flagella

    PubMed Central

    Mukherjee, Shatanik; Jansen, Vera; Jikeli, Jan F; Hamzeh, Hussein; Alvarez, Luis; Dombrowski, Marco; Balbach, Melanie; Strünker, Timo; Seifert, Reinhard; Kaupp, U Benjamin; Wachten, Dagmar

    2016-01-01

    The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca2+, basal SACY activity is suppressed by Ca2+. Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo. DOI: http://dx.doi.org/10.7554/eLife.14052.001 PMID:27003291

  4. Novel cAMP signalling paradigms: therapeutic implications for airway disease.

    PubMed

    Billington, Charlotte K; Hall, Ian P

    2012-05-01

    Since its discovery over 50 years ago, cAMP has been the archetypal second messenger introducing students to the concept of cell signalling at the simplest level. As explored in this review, however, there are many more facets to cAMP signalling than the path from Gs-coupled receptor to adenylyl cyclase (AC) to cAMP to PKA to biological effect. After a brief description of this canonical cAMP signalling pathway, a snapshot is provided of the novel paradigms of cAMP signalling. As in the airway the cAMP pathway relays the major bronchorelaxant signal and as such is the target for frontline therapy for asthma and COPD, particular emphasis is given to airway disease and therapy. Areas discussed include biased agonism, continued signalling following internalization, modulation of cAMP by AC, control of cAMP degradation, cAMP and calcium crosstalk, Epac-mediated signalling and finally the implications of altered genotypes will be considered. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.

  5. Antibacterial Mode of Action of Ib-AMP1 Against Escherichia coli O157:H7.

    PubMed

    Wu, Wen-Hsuan; Di, Rong; Matthews, Karl R

    2013-06-01

    Continual occurrence of foodborne outbreaks, along with the increase in antibiotic resistance which burdens clinical treatments, has urged scientists to search for other potential promising antimicrobial agents. Antimicrobial peptides are emerging as one of the potential alternatives. The mode of action of a given AMP is critical and essential for future application; however, it is still not completely known for many of these compounds. Ib-AMP1 is a plant-derived AMP, purified from seeds of Impatiens balsamina and has been shown to exert antibacterial and antifungal activity at the micromolar level. A study had shown that the therapeutic index of Ib-AMP1 against eight human pathogens is 23.5. The objective of the present study was to determine the in vivo mode of action of Ib-AMP1 against Escherichia coli O157:H7. A concentration-dependent effect of Ib-AMP1 on the E. coli O157:H7 cell membrane occurred. Ib-AMP1 treatments resulted in efflux of K(+) and ATP, suggesting pores of sufficient size to allow efflux of large molecules. Ib-AMP1 at sublethal concentrations exerts a greater effect at the intracellular level. In contrast, Ib-AMP1 at a lethal concentration permeabilizes cell membranes and may directly or indirectly inhibit intracellular macromolecule synthesis. Collectively, results of this study suggest Ib-AMP1 is bactericidal interfering within outer and inner membrane integrity permitting efflux of ATP and interfering with intracellular biosynthesis of DNA, RNA and protein.

  6. A novel biosensor to study cAMP dynamics in cilia and flagella.

    PubMed

    Mukherjee, Shatanik; Jansen, Vera; Jikeli, Jan F; Hamzeh, Hussein; Alvarez, Luis; Dombrowski, Marco; Balbach, Melanie; Strünker, Timo; Seifert, Reinhard; Kaupp, U Benjamin; Wachten, Dagmar

    2016-03-22

    The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca(2+), basal SACY activity is suppressed by Ca(2+). Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo.

  7. Leveraging family-specific signatures for AMP discovery and high-throughput annotation

    PubMed Central

    Waghu, Faiza Hanif; Barai, Ram Shankar; Idicula-Thomas, Susan

    2016-01-01

    Antimicrobial peptides (AMPs) are diverse, biologically active, essential components of the innate immune system. As compared to conventional antibiotics, AMPs exhibit broad spectrum antimicrobial activity, reduced toxicity and reduced microbial resistance. They are widely researched for their therapeutic potential, especially against multi-drug resistant pathogens. AMPs are known to have family-specific sequence composition, which can be mined for their discovery and rational design. Here, we present a detailed family-based study on AMP families. The study involved the use of sequence signatures represented by patterns and hidden Markov models (HMMs) present in experimentally studied AMPs to identify novel AMPs. Along with AMPs, peptides hitherto lacking antimicrobial annotation were also retrieved and wet-lab studies on randomly selected sequences proved their antimicrobial activity against Escherichia coli. CAMPSign, a webserver has been created for researchers to effortlessly exploit the use of AMP family signatures for identification of AMPs. The webserver is available online at www.campsign.bicnirrh.res.in. In this work, we demonstrate an optimised and experimentally validated protocol along with a freely available webserver that uses family-based sequence signatures for accelerated discovery of novel AMPs. PMID:27089856

  8. A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers.

    PubMed

    Astafieva, A A; Rogozhin, Eugene A; Andreev, Yaroslav A; Odintsova, T I; Kozlov, S A; Grishin, Eugene V; Egorov, Tsezi A

    2013-09-01

    A novel peptide named ToAMP4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, and RP-HPLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids, and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 does not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4, it was successfully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4-mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and functional diversity of AMPs in plants.

  9. Cardiac myocyte–secreted cAMP exerts paracrine action via adenosine receptor activation

    PubMed Central

    Sassi, Yassine; Ahles, Andrea; Truong, Dong-Jiunn Jeffery; Baqi, Younis; Lee, Sang-Yong; Husse, Britta; Hulot, Jean-Sébastien; Foinquinos, Ariana; Thum, Thomas; Müller, Christa E.; Dendorfer, Andreas; Laggerbauer, Bernhard; Engelhardt, Stefan

    2014-01-01

    Acute stimulation of cardiac β-adrenoceptors is crucial to increasing cardiac function under stress; however, sustained β-adrenergic stimulation has been implicated in pathological myocardial remodeling and heart failure. Here, we have demonstrated that export of cAMP from cardiac myocytes is an intrinsic cardioprotective mechanism in response to cardiac stress. We report that infusion of cAMP into mice averted myocardial hypertrophy and fibrosis in a disease model of cardiac pressure overload. The protective effect of exogenous cAMP required adenosine receptor signaling. This observation led to the identification of a potent paracrine mechanism that is dependent on secreted cAMP. Specifically, FRET-based imaging of cAMP formation in primary cells and in myocardial tissue from murine hearts revealed that cardiomyocytes depend on the transporter ABCC4 to export cAMP as an extracellular signal. Extracellular cAMP, through its metabolite adenosine, reduced cardiomyocyte cAMP formation and hypertrophy by activating A1 adenosine receptors while delivering an antifibrotic signal to cardiac fibroblasts by A2 adenosine receptor activation. Together, our data reveal a paracrine role for secreted cAMP in intercellular signaling in the myocardium, and we postulate that secreted cAMP may also constitute an important signal in other tissues. PMID:25401477

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

    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

  12. AmpH, a Bifunctional dd-Endopeptidase and dd-Carboxypeptidase of Escherichia coli▿

    PubMed Central

    González-Leiza, Silvia M.; de Pedro, Miguel A.; Ayala, Juan A.

    2011-01-01

    In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display dd-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related to AmpC β-lactamases. AmpH has been associated with PG recycling, although its enzymatic activity remained uncharacterized until now. Construction and purification of His-tagged AmpH from E. coli permitted a detailed study of its enzymatic properties. The N-terminal export signal of AmpH is processed, but the protein remains membrane associated. The PBP nature of AmpH was demonstrated by its ability to bind the β-lactams Bocillin FL (a fluorescent penicillin) and cefmetazole. In vitro assays with AmpH and specific muropeptides demonstrated that AmpH is a bifunctional dd–endopeptidase and dd-carboxypeptidase. Indeed, the enzyme cleaved the cross-linked dimers tetrapentapeptide (D45) and tetratetrapeptide (D44) with efficiencies (kcat/Km) of 1,200 M−1 s−1 and 670 M−1 s−1, respectively, and removed the terminal d-alanine from muropeptides with a C-terminal d-Ala-d-Ala dipeptide. Both dd-peptidase activities were inhibited by 40 μM cefmetazole. AmpH also displayed a weak β-lactamase activity for nitrocefin of 1.4 × 10−3 nmol/μg protein/min, 1/1,000 the rate obtained for AmpC under the same conditions. AmpH was also active on purified sacculi, exhibiting the bifunctional character that was seen with pure muropeptides. The wide substrate spectrum of the dd-peptidase activities associated with AmpH supports a role for this protein in PG remodeling or recycling. PMID:22001512

  13. Foundational development of an advanced nuclear reactor integrated safety code.

    SciTech Connect

    Clarno, Kevin; Lorber, Alfred Abraham; Pryor, Richard J.; Spotz, William F.; Schmidt, Rodney Cannon; Belcourt, Kenneth; Hooper, Russell Warren; Humphries, Larry LaRon

    2010-02-01

    This report describes the activities and results of a Sandia LDRD project whose objective was to develop and demonstrate foundational aspects of a next-generation nuclear reactor safety code that leverages advanced computational technology. The project scope was directed towards the systems-level modeling and simulation of an advanced, sodium cooled fast reactor, but the approach developed has a more general applicability. The major accomplishments of the LDRD are centered around the following two activities. (1) The development and testing of LIME, a Lightweight Integrating Multi-physics Environment for coupling codes that is designed to enable both 'legacy' and 'new' physics codes to be combined and strongly coupled using advanced nonlinear solution methods. (2) The development and initial demonstration of BRISC, a prototype next-generation nuclear reactor integrated safety code. BRISC leverages LIME to tightly couple the physics models in several different codes (written in a variety of languages) into one integrated package for simulating accident scenarios in a liquid sodium cooled 'burner' nuclear reactor. Other activities and accomplishments of the LDRD include (a) further development, application and demonstration of the 'non-linear elimination' strategy to enable physics codes that do not provide residuals to be incorporated into LIME, (b) significant extensions of the RIO CFD code capabilities, (c) complex 3D solid modeling and meshing of major fast reactor components and regions, and (d) an approach for multi-physics coupling across non-conformal mesh interfaces.

  14. AmpG Inactivation Restores Susceptibility of Pan-β-Lactam-Resistant Pseudomonas aeruginosa Clinical Strains▿

    PubMed Central

    Zamorano, Laura; Reeve, Thomas M.; Juan, Carlos; Moyá, Bartolomé; Cabot, Gabriel; Vocadlo, David J.; Mark, Brian L.; Oliver, Antonio

    2011-01-01

    Constitutive AmpC hyperproduction is the most frequent mechanism of resistance to the weak AmpC inducers antipseudomonal penicillins and cephalosporins. Previously, we demonstrated that inhibition of the β-N-acetylglucosaminidase NagZ prevents and reverts this mechanism of resistance, which is caused by ampD and/or dacB (PBP4) mutations in Pseudomonas aeruginosa. In this work, we compared NagZ with a second candidate target, the AmpG permease for GlcNAc-1,6-anhydromuropeptides, for their ability to block AmpC expression pathways. Inactivation of nagZ or ampG fully restored the susceptibility and basal ampC expression of ampD or dacB laboratory mutants and impaired the emergence of one-step ceftazidime-resistant mutants in population analysis experiments. Nevertheless, only ampG inactivation fully blocked ampC induction, reducing the MICs of the potent AmpC inducer imipenem from 2 to 0.38 μg/ml. Moreover, through population analysis and characterization of laboratory mutants, we showed that ampG inactivation minimized the impact on resistance of the carbapenem porin OprD, reducing the MIC of imipenem for a PAO1 OprD mutant from >32 to 0.5 μg/ml. AmpG and NagZ targets were additionally evaluated in three clinical isolates that are pan-β-lactam resistant due to AmpC hyperproduction, OprD inactivation, and overexpression of several efflux pumps. A marked increase in susceptibility to ceftazidime and piperacillin-tazobactam was observed in both cases, while only ampG inactivation fully restored wild-type imipenem susceptibility. Susceptibility to meropenem, cefepime, and aztreonam was also enhanced, although to a lower extent due to the high impact of efflux pumps on the activity of these antibiotics. Thus, our results suggest that development of small-molecule inhibitors of AmpG could provide an excellent strategy to overcome the relevant mechanisms of resistance (OprD inactivation plus AmpC induction) to imipenem, the only currently available β-lactam not

  15. Changes of concentration of cyclic AMP in rat brain and plasma in the clinical death model.

    PubMed

    Kapuściński, A

    1991-01-01

    In the experimental model of clinical death in rats (Korpachev et al. 1982) cyclic AMP concentrations were evaluated in the brain and plasma at the end of 5-min clinical death, and 5, 15, 30, 60 and 120 min after resuscitation. The cAMP 125I assay system has been used. At the end of clinical death the cAMP level decreased in the brain with normalization 15 min after resuscitation; the second decrease of the cAMP level was observed 30 min post resuscitation with normalization in later periods. In the plasma cAMP concentration did not change at the end of clinical death, followed by a significant increase 5 min after resuscitation. Later the level of plasma cAMP decreased being still above the control value after 2 hours. The possible role of endogenous catecholamines stimulation on adenylate cyclase activity is discussed.

  16. Complementation of growth defect in an ampC deletion mutant of Escherichia coli.

    PubMed

    Bishop, R E; Weiner, J H

    1993-12-15

    beta-Lactamase genes of class-A (Rtem) and class-C (ampC) were placed under control of an inducible tac-promoter and expressed in Escherichia coli. Expression of RTEM had no observable effect on the growth properties of E. coli strains HB101 (ampC+) or MI1443 (delta ampC). E. coli MI1443 exhibited a decline in growth rate at mid-exponential phase which could be delayed by expression of AmpC at early-exponential phase. AmpC expression otherwise inhibited growth, particularly during the transition into exponential phase where growth was prevented altogether. We suggest that the AmpC beta-lactamase, but not RTEM, may have an additional cellular function as a peptidoglycan hydrolase.

  17. Cyclic AMP-dependent phosphorylation in the control of biotransformation in the liver.

    PubMed

    Bánhegyi, G; Garzó, T; Mészáros, G; Faragó, A; Antoni, F; Mandl, J

    1988-03-01

    The possibility of a short-term cAMP-dependent regulation of mixed-function oxidation and of glucuronide formation was investigated in isolated mouse hepatocytes and in mouse liver microsomal membranes. N6, O2-dibutyryl cAMP (in accordance with its increasing effect on gluconeogenesis) decreased aminopyrine oxidation and p-nitrophenol conjugation in isolated hepatocytes, while the phenolphthalein conjugation remained unaltered. Similar to dibutyryl cAMP the Ca2+ ionophore A 23187 also decreased aminopyrine oxidation. In cell-free systems the phosphorylation of isolated microsomal membranes by the exogenous cAMP-dependent protein kinase was inhibitory on aminopyrine oxidation and p-nitrophenol glucuronide formation but aniline oxidation and phenolphthalein glucuronidation were not affected. The correlation between the negative cAMP-dependent control of certain processes of biotransformation and the positive cAMP-dependent regulation of gluconeogenesis is discussed.

  18. cAMP signalling in the normal and tumorigenic pituitary gland.

    PubMed

    Formosa, R; Vassallo, J

    2014-07-05

    cAMP signalling plays a key role in the normal physiology of the pituitary gland, regulating cellular growth and proliferation, hormone production and release. Deregulation of the cAMP signalling pathway has been reported to be a common occurrence in pituitary tumorigenesis. Several mechanisms have been implicated including somatic mutations, gene-gene interactions and gene-environmental interactions. Somatic mutations in G-proteins and protein kinases directly alter cAMP signalling, while malfunctioning of other signalling pathways such as the Raf/MAPK/ERK, PI3K/Akt/mTOR and Wnt pathways which normally interact with the cAMP pathway may mediate indirect effects on cAMP and varying downstream effectors. The aryl hydrocarbon receptor signalling pathway has been implicated in pituitary tumorigenesis and we review its role in general and specifically in relation to cAMP de-regulation.

  19. Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?

    PubMed Central

    Pérez-Gallego, Marcelo; Torrens, Gabriel; Castillo-Vera, Jane; Moya, Bartolomé; Zamorano, Laura; Hultenby, Kjell; Albertí, Sebastián; Mellroth, Peter; Henriques-Normark, Birgitta; Normark, Staffan

    2016-01-01

    ABSTRACT Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa, a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD, encoding an N-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels of ampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis. PMID:27795406

  20. Dynamics of β-adrenergic/cAMP signaling and morphological changes in cultured astrocytes.

    PubMed

    Vardjan, Nina; Kreft, Marko; Zorec, Robert

    2014-04-01

    The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease.

  1. Is This Op-Amp Any Good?: Lab-Built Checker Removes All Doubt!

    ERIC Educational Resources Information Center

    Harman, Charles

    2007-01-01

    Electronics instructors and students find it very helpful to be able to check an operational amplifier at the proto-board stage. Most students lack the experience or knowledge that it takes to recognize whether an op-amp is operating normally or not. This article discusses a handy op-amp checker that allows one to check and/or test op-amps at the…

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

    NASA Astrophysics Data System (ADS)

    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.

  3. Nanomolar Inhibitors of AmpC [beta]-Lactamase

    SciTech Connect

    Morandi, Federica; Caselli, Emilia; Morandi, Stefania; Focia, Pamela J.; Blazquez, Jesus; Shoichet, Brian K.; Prati, Fabio

    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.

  4. cAMP-COUPLED RIBOFLAVIN TRAFFICKING IN PLACENTAL TROPHOBLASTS

    PubMed Central

    D’Souza, Vanessa M.; Foraker, Amy B.; Free, R. Benjamin; Ray, Abhijit; Shapiro, Paul S.; Swaan, Peter W.

    2008-01-01

    Riboflavin (RF, vitamin B2), an essential micronutrient central to cellular metabolism through formation of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors, is internalized, at least in part, via a proposed receptor-mediated endocytic (RME) process. The purpose of this study was to delineate the cellular RF distribution using human placental trophoblasts, and evaluate the regulatory role of cAMP in this process. Subcellular fractionation and 3-D confocal microscopy analyses were carried out to define the RF accumulation profile. Biochemical assays evaluating the cAMP dependence of this pathway were also performed. The present study records an intracellular RF distribution pattern that shows dynamic accumulation of the ligand predominantly, to the endosomal and lysosomal compartments and to a lesser extent to the Golgi and mitochondria. In contrast, transferrin (TF) colocalizes rapidly within endosomes with minimal accumulation in the other organelles. Temporal and spatial distribution of RF and TF colocalized with unique markers of the endocytic machinery provide added morphological evidence in support of the RME process with ultimate translocation to the mitochondrial domain. Colocalized staining with the Golgi also suggests a possible recycling or exocytic mechanism for this ligand. Furthermore, this study demonstrates cAMP regulation of the putative ligand-bound RF receptor and its association into endocytic vesicles. Delineating the dynamics of the process governing cellular RF homeostasis presents an untapped resource that can be further exploited to improve our current understanding of nutritional biology and fetal growth and development, and perhaps target the endogenous system to develop novel therapeutic approaches. PMID:16681382

  5. Amp-hour counting control for PV hybrid power systems

    SciTech Connect

    Hund, T.D.; Thompson, B.

    1997-06-01

    The performance of an amp-hour (Ah) counting battery charge control algorithm has been defined and tested using the Digital Solar Technologies MPR-9400 microprocessor based PV hybrid charge controller. This work included extensive field testing of the charge algorithm on flooded lead-antimony and valve regulated lead-acid (VRLA) batteries. The test results after one-year have demonstrated that PV charge utilization, battery charge control, and battery state of charge (SOC) has been significantly improved by providing maximum charge to the batteries while limiting battery overcharge to manufacturers specifications during variable solar resource and load periods.

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

    PubMed

    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.

  7. Structural and Functional Characterization of Pseudomonas aeruginosa Global Regulator AmpR

    PubMed Central

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

    2014-01-01

    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 σ54 and σ70 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

  8. Genotypic Identification of AmpC β-Lactamases Production in Gram-Negative Bacilli Isolates

    PubMed Central

    Wassef, Mona; Behiry, Iman; Younan, Mariam; El Guindy, Nancy; Mostafa, Sally; Abada, Emad

    2014-01-01

    Background: AmpC type β-lactamases are commonly isolated from extended-spectrum Cephalosporin-resistant Gram-negative bacteria. Also, resistance appeared in bacterial species not naturally producing AmpC enzymes. Therefore, a standard test for the detection of the plasmid-mediated AmpC enzyme and new breakpoints for extended spectrum Cephalosporins are urgently necessary. Objectives: To detect plasmid and chromosomal mediated AmpC-β-lactamases in Gram negative bacteria in community and hospital acquired infections. Materials and Methods: 1073 Gram negative clinical isolates were identified by the conventional methods and were screened for AmpC production using Cefoxitin discs. Confirmatory phenotypic identifications were done for the Cefoxitin-resistant isolates using Boronic Acid for combined and double disc synergy tests, Cloxacillin based double disc synergy test, and induction tests. The genotypic identification of plasmid-mediated AmpC was done using multiplex PCR. ESBL production was also screened by discs of Ceftazidime and Cefotaxime with and without Clavulanic Acid (10 μg). Results: The AmpC-producing isolates among all identified Gram negative bacilli were 5.8% (62/1073) as detected by screening disc diffusion methods, where 72% were positive for AmpC by combined disc method (Cefotetan and Boronic Acid), 56.5% were positive by each of Boronic Acid and Cloxacillin double disc synergy tests, 35.5% were positive by the induction test, and 25.8% were plasmid-mediated AmpC β-lactamase producers by the multiplex PCR. Plasmid-mediated AmpC genes retrieved, belonged to the families (MOX, FOX, EBC and CIT). ESBL producers were found in 26 (41.9%) isolates, 15 (57%) of which also produced AmpC. Isolates caused hospital acquired infections were (53/62); of which (39/62) were AmpC producers. While only (8/62) of the isolates caused community-acquired infections, were AmpC producers, and (1.6%) (1/62) were non AmpC producer. Conclusions: The AmpC

  9. cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

    SciTech Connect

    Ghayor, Chafik; Ehrbar, Martin; Miguel, Blanca San; Graetz, Klaus W.; Weber, Franz E.

    2009-04-03

    Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitor of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.

  10. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase

    SciTech Connect

    Townley,R.; Shapiro, L.

    2007-01-01

    The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular adenosine triphosphate (ATP) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Angstrom resolution for ATP- and AMP-bound forms of a core {alpha}{beta}{gamma} adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the {gamma} subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

  11. Regulating the ubiquitin/proteasome pathway via cAMP-signaling: neuroprotective potential

    PubMed Central

    Huang, He; Wang, Hu; Figueiredo-Pereira, Maria E.

    2013-01-01

    The cAMP-signaling pathway has been under intensive investigation for decades. It is a wonder that such a small simple molecule like cAMP can modulate a vast number of diverse processes in different types of cells. The ubiquitous involvement of cAMP-signaling in a variety of cellular events requires tight spatial and temporal control of its generation, propagation, compartmentalization, and elimination. Among the various steps of the cAMP-signaling pathway, G-protein coupled receptors, adenylate cyclases, phosphodiesterases, the two major cAMP targets, i.e. protein kinase A and exchange protein activated by cAMP, as well as the A-kinase anchoring proteins, are potential targets for drug development. Herein we review the recent progress on the regulation and manipulation of different steps of the cAMP-signaling pathway. We end by focusing on the emerging role of cAMP-signaling in modulating protein degradation via the ubiquitin/proteasome pathway. New discoveries on the regulation of the ubiquitin/proteasome pathway by cAMP-signaling support the development of new therapeutic approaches to prevent proteotoxicity in chronic neurodegenerative disorders and other human disease conditions associated with impaired protein turnover by the ubiquitin/proteasome pathway and the accumulation of ubiquitin-protein aggregates. PMID:23686612

  12. Ethanol-induced loss of brain cyclic AMP binding proteins: correlation with growth suppression

    SciTech Connect

    Pennington, S.; Kalmus, G.

    1987-05-01

    Brain hypoplasia secondary to maternal ethanol consumption is a common fetal defect observed in all models of fetal alcohol syndrome. The molecular mechanism by which ethanol inhibits growth is unknown but has been hypothesized to involve ethanol-induced changes in the activity of cyclic-AMP stimulated protein kinase. Acute and chronic alcohol exposure elevate cyclic AMP level in many tissues, including brain. This increase in cyclic AMP should increase the phosphorylating activity of kinase by increasing the amount of dissociated (active) kinase catalytic subunit. In 7-day embryonic chick brains, ethanol-induced growth suppression was correlated with increased brain cyclic AMP content but neither basal nor cyclic AMP stimulated kinase catalytic activity was increased. However, the levels of cyclic AMP binding protein (kinase regulatory subunit) were significantly lowered by ethanol exposure. Measured as either /sup 3/H cyclic AMP binding or as 8-azido cyclic AM/sup 32/P labeling, ethanol-exposed brains had significantly less cyclic AMP binding activity (51 +/- 14 versus 29 +/- 10 units/..mu..g protein for 8-azido cyclic AMP binding). These findings suggest that ethanol's effect on kinase activity may involve more than ethanol-induced activation of adenylate cyclase.

  13. The binding of tyrosinyl-5'-AMP to tyrosyl-tRNA synthetase (E.coli).

    PubMed Central

    Grosse, F; Krauss, G; Kownatzki, R; Maass, G

    1979-01-01

    The binding between tyrosyl-tRNA synthetase (E.coli) and the alkylanalogue of the aminoacyladenylate, tyrosinyl-5'-AMP, has been investigated by fluorescence titrations and rapid mixing experiments. Tyrosyl-tRNA synthetase has two equivalent and independent binding sites for tyrosinyl-5'-AMP. The intrinsic binding constant is 4 x 10(7)M-1. The binding sites for tRNATyr and tyrosinyl-5'-AMP are independent of each other, the anticooperative mode of tRNA binding being preserved in the presence of tyrosinyl-5-AMP. PMID:377229

  14. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

    PubMed

    Zhou, Zhiwen; Tanaka, Kenji F; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-22

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

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

    PubMed

    Fayet, G; Aouani, A; Hovsépian, S

    1986-01-06

    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.

  16. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein.

    PubMed

    Popovych, Nataliya; Tzeng, Shiou-Ru; Tonelli, Marco; Ebright, Richard H; Kalodimos, Charalampos G

    2009-04-28

    The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.

  17. Enhanced cAMP accumulation by a phorbol ester in cerebral cortical cells

    SciTech Connect

    Beeler, J.F.; Davis, C.W.

    1987-05-01

    Phorbol 12-myristate-13-acetate (PMA) was found to be selective in its ability to alter cAMP accumulations in cultured rat cerebral cortical cells. Basal levels of cAMP in cultured neuronal and nonneuronal cells preincubated in the absence or presence of PMA were 14 pmol/mg protein and 16 pmol/mg protein, respectively. Adenosine increased cAMP levels in a dose-dependent manner. cAMP accumulation in response to low concentrations of adenosine was not significantly altered by pretreatment with PMA but marked potentiation of adenosine elicited accumulations was observed at 10 and 100 ..mu..M adenosine. Longer preincubation with PMA resulted in a decreased ability of PMA to enhance adenosine elicited accumulations of cAMP. PMA did not significantly alter cAMP accumulation by forskolin (FOR) and enhanced norepinephrine stimulated cAMP by only 2-fold. For similarly potentiated adenosine/sub 2/ (A/sub 2/)- receptor elicited accumulation of cAMP which could be further enhanced by PMA. These results suggest that the effects of the phorbol ester are more specific for potentiating adenosine stimulated cAMP accumulation and may occur as a result of a more efficient coupling between the A/sub 2/-receptor, N-protein and adenylate cyclase.

  18. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis

    PubMed Central

    Zhou, Zhiwen; Tanaka, Kenji F.; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  19. A-kinase anchoring proteins: cAMP compartmentalization in neurodegenerative and obstructive pulmonary diseases

    PubMed Central

    Poppinga, W J; Muñoz-Llancao, P; González-Billault, C; Schmidt, M

    2014-01-01

    The universal second messenger cAMP is generated upon stimulation of Gs protein-coupled receptors, such as the β2-adreneoceptor, and leads to the activation of PKA, the major cAMP effector protein. PKA oscillates between an on and off state and thereby regulates a plethora of distinct biological responses. The broad activation pattern of PKA and its contribution to several distinct cellular functions lead to the introduction of the concept of compartmentalization of cAMP. A-kinase anchoring proteins (AKAPs) are of central importance due to their unique ability to directly and/or indirectly interact with proteins that either determine the cellular content of cAMP, such as β2-adrenoceptors, ACs and PDEs, or are regulated by cAMP such as the exchange protein directly activated by cAMP. We report on lessons learned from neurons indicating that maintenance of cAMP compartmentalization by AKAP5 is linked to neurotransmission, learning and memory. Disturbance of cAMP compartments seem to be linked to neurodegenerative disease including Alzheimer's disease. We translate this knowledge to compartmentalized cAMP signalling in the lung. Next to AKAP5, we focus here on AKAP12 and Ezrin (AKAP78). These topics will be highlighted in the context of the development of novel pharmacological interventions to tackle AKAP-dependent compartmentalization. PMID:25132049

  20. Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli.

    PubMed Central

    Phillips, A T; Egan, R M; Lewis, B

    1978-01-01

    To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine. PMID:211115

  1. Molecular imprinting of AMP by an ionic-noncovalent dual approach.

    PubMed

    Breton, Florent; Delépée, Raphaël; Agrofoglio, Luigi A

    2009-10-01

    In order to mimic recognition properties of adenylate kinase, molecularly imprinted polymers (MIPs) were prepared for adenosine 5'-monophosphate (AMP), a substrate of the enzyme. Different functional monomers interacting with the phosphate moiety were tested, and the MIP giving the best specific binding of AMP was composed with one equivalent of 2-(dimethylamino)ethyl methacrylate and ten equivalents of acrylamide compared to AMP. Packed into solid phase cartridge, this polymer showed similar characteristics than the enzyme, since it was specific for AMP toward other nucleotides.

  2. The concentration of cyclic AMP and the activity of cyclic AMP dependent protein kinase and an inhibitor in the adipose tissue of rats fed lard or glucose diets.

    PubMed

    Jackowski, M M; Tepperman, H M; Tepperman, J

    1978-08-01

    Measurements of tissue cyclic AMP (cAMP) concentration, the activity of cAMP-dependent protein kinase and the level of the enzyme's thermostable, macromolecular inhibitor were made on preparations of rat epididymal fat pad from animals fed high fat or high carbohydrate diets. The cAMP concentration from rats adapted to a high lard diet for 14-15 days was 153 +/- 17.8 pmoles/mg protein as opposed to 76 +/- 6.0 found with high glucose diet. No significant difference in total cAMP-dependent protein kinase activity was observed among rats fed high glucose, high lard or laboratory chow, although the enzyme's activity ratio (-cAMP)(+cAMP) was significantly elevated with lard feeding (0.49 +/- 0.02) as opposed to glucose feeding (0.43 +/- 0.01). Crude preparations from lard and glucose fed animals were equivalent in inhibitory activity when tested with enzyme from chow fed animals. Agarose column chromatography separated holoenzyme and C subunit forms of the protein kinase when 500 mM NaCl was present in the elution buffer. Absence of the salt allowed subunit reassociation to occur. Direct addition of NaCl greater than or equal to 75 mM significantly inhibited protein kinase activity. The results indicate that the adipose tissue of rats fed a high lard diet has a higher concentration of cAMP and an increased protein kinase activity ratio than tissue from rats fed a fat free, high glucose diet. Total cAMP-dependent protein kinase activity and the level of a thermostable macromolecular inhibitor remained unchanged.

  3. Reanalyzing the Ampère-Maxwell Law

    NASA Astrophysics Data System (ADS)

    Hill, S. Eric

    2011-09-01

    In a recent TPT article, I addressed a common miscommunication about Faraday's law, namely, that introductory texts often say the law expresses a causal relationship between the magnetic fields time variation and the electric fields circulation. In that article, I demonstrated that these field behaviors share a common cause in a time-varying current density. From that, many readers may have rightly guessed at a symmetric conclusion: while the Ampère-Maxwell law is commonly said to express a causal relation between the electric fields time variation and the magnetic fields circulation, these field behaviors share a distinct, common cause. Together, Faraday's law and the Ampère-Maxwell law constitute half of Maxwell's laws that form a foundation for almost all of electricity and magnetism. By misrepresenting these two laws, introductory texts not only present students with unnecessary conceptual hurdles early in their physics educations but also leave them with enduring misunderstandings about the very foundation of electricity and magnetism. Fortunately, compared to what is commonly taught, the actual cause of these field variations is conceptually simpler and more consistent with what the students will have already learned in the introductory texts' own earlier chapters.

  4. Solution structure of the cAMP-dependent protein kinase

    SciTech Connect

    Trewhella, J.; Olah, G.A.; Walsh, D.A.; Mitchell, R.D.

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project as Los Alamos National Laboratory (LANL). Protein phosphorylation is well established as one of the most important mechanisms of signal transduction and cellular regulation. Two of the key enzymes that catalyze these phosphorylation reactions are the cAMP- (PKA) and cGMP- (PKG) dependent protein kinases. PKA has served as the prototypic model of this class of enzymes that now comprises in excess of 300 phylogenetically related proteins. A large number of these protein kinases are critical for the regulation of cell function and a full analysis of their similarities and differences is essential to understand their diverse physiological roles. The cAMP-dependent protein kinase has the subunit structure R2C2, in which C and R refer to the catalytic and regulatory subunits, respectively. The cGMP-dependent protein kinase (PKG) is highly homologous to PKA but is distinguished from it by having the regulatory and catalytic domains on a contiguous polypeptide. The studies described here use small-angle scattering and Fourier Transform InfraRed (FTIR) spectroscopy to study domain movements and conformational changes in these enzymes in different functional states in order to elucidate the molecular bases for the regulation of their activities.

  5. Non-Enzymatic Oligomerization of 3', 5' Cyclic AMP.

    PubMed

    Costanzo, Giovanna; Pino, Samanta; Timperio, Anna Maria; Šponer, Judit E; Šponer, Jiří; Nováková, Olga; Šedo, Ondrej; Zdráhal, Zbyněk; Di Mauro, Ernesto

    2016-01-01

    Recent studies illustrate that short oligonucleotide sequences can be easily produced from nucleotide precursors in a template-free non-enzymatic way under dehydrating conditions, i.e. using essentially dry materials. Here we report that 3',5' cyclic AMP may also serve as a substrate of the reaction, which proceeds under moderate conditions yet with a lower efficiency than the previously reported oligomerization of 3',5' cyclic GMP. Optimally the oligomerization requires (i) a temperature of 80°C, (ii) a neutral to alkaline environment and (iii) a time on the order of weeks. Differences in the yield and required reaction conditions of the oligomerizations utilizing 3',5' cGMP and cAMP are discussed in terms of the crystal structures of the compounds. Polymerization of 3',5' cyclic nucleotides, whose paramount relevance in a prebiotic chemistry context has been widely accepted for decades, supports the possibility that the origin of extant genetic materials might have followed a direct uninterrupted path since its very beginning, starting from non-elaborately pre-activated monomer compounds and simple reactions.

  6. Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

    PubMed Central

    Škrnjug, Ivana

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity – a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines. PMID:25295996

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

    PubMed

    Paramonov, Valeriy M; Mamaeva, Veronika; Sahlgren, Cecilia; Rivero-Müller, Adolfo

    2015-01-01

    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. Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

    PubMed Central

    Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

    2016-01-01

    High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

  9. cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

    PubMed

    Li, Xiaoying; Tao, Hua; Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

  10. cAMP Signaling Prevents Podocyte Apoptosis via Activation of Protein Kinase A and Mitochondrial Fusion

    PubMed Central

    Xie, Kewei; Ni, Zhaohui; Yan, Yucheng; Wei, Kai; Chuang, Peter Y.; He, John Cijiang; Gu, Leyi

    2014-01-01

    Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP. PMID:24642777

  11. A reassessment of the modulatory role of cyclic AMP in catecholamine secretion by chromaffin cells.

    PubMed Central

    Parramón, M; González, M P; Oset-Gasque, M J

    1995-01-01

    1. The role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in the regulation of catecholamine (CA) secretion in chromaffin cells remains equivocal from previous studies. 2. In the present study the effect of this cyclic nucleotide on basal CA secretion, as well as on intracellular calcium and membrane potential has been examined. 3. Forskolin and the permeable cyclic AMP analogue, 8-(4-chlorphenylthio)-adenosine-3'-5' monophosphate cyclic (pClpcAMP), increased basal CA secretion in a dose-dependent manner. The EC50s were 0.43 +/- 0.10 microM for forskolin and 39 +/- 9 microM for pClpcAMP. Other agonists with adenylate cyclase activity such as stimulants of adenosine receptors, beta-adrenoceptors, GABAB receptors and intestinal vasoactive peptide (VIP), also increased basal CA secretion in a highly significant manner. However, when they were added together with forskolin, CA secretion was not affected although an additive increase in cyclic AMP levels was produced. 4. Statistical analysis of the correlation between cyclic AMP levels and CA secretion evoked by these cyclic AMP increasing compounds showed that a significant direct correlation between both parameters existed only when low levels of cyclic AMP were produced by secretagogue stimulation. When the increase in intracellular cyclic AMP concentrations exceeded approximately 8 times the basal cyclic AMP levels the correlation was not significant. These results indicate a dual dose-dependent effect of cyclic AMP on basal CA secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7881750

  12. Dose and Chemical Modification Considerations for Continuous Cyclic AMP Analog Delivery to the Injured CNS

    PubMed Central

    Fouad, Karim; Ghosh, Mousumi; Vavrek, Romana; Tse, Arthur D.

    2009-01-01

    Abstract In this investigation, two cell-permeable synthetic analogs of cAMP, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, which are widely used to elevate intracellular cAMP levels under experimental conditions, were investigated for their ability to dose-dependently improve histological and functional outcomes following continuous delivery in two models of incomplete spinal cord injury (SCI). The cAMP analogs were delivered via osmotic minipumps at 1–250 mM through an indwelling cortical cannula or by intrathecal infusion for up to 4 weeks after either a T8 unilateral over-hemisection or a C2-3 dorsolateral quadrant lesion, respectively. In both SCI models, continuous db-cAMP delivery was associated with histopathological changes that included sporadic micro-hemorrhage formation and cavitation, enhanced macrophage infiltration and tissue damage at regions beyond the immediate application site; no deleterious or beneficial effect of agent delivery was observed at the spinal injury site. Furthermore, these changes were accompanied by pronounced behavioral deficits that included an absence of progressive locomotor recovery, increased extensor tone, paralysis, and sensory abnormalities. These deleterious effects were not observed in saline-treated animals, in animals in which the db-cAMP dose did not exceed 1 mM, or in those animals that received a high dose (250 mM) of the alternative cAMP analog, 8-bromo-cAMP. These results demonstrate that, for continuous intraparenchymal or intrathecal administration of cAMP analogs for the study of biological or therapeutic effects within the central nervous system (CNS), consideration of the effective concentration applied as well as the potential toxicity of chemical moieties on the parent molecule and/or their activity needs to be taken into account. PMID:19397425

  13. SHC1 sensitizes cancer cells to the 8-Cl-cAMP treatment.

    PubMed

    Choi, Ki Young; Cho, Young Jun; Kim, Jeong Seon; Ahn, Young-Ho; Hong, Seung Hwan

    2015-08-07

    8-Chloro-cyclic AMP (8-Cl-cAMP) is a cyclic AMP analog that induces growth inhibition and apoptosis in a broad spectrum of cancer cells. Previously, we found that 8-Cl-cAMP-induced growth inhibition is mediated by AMP-activated protein kinase (AMPK) as well as p38 mitogen-activated protein kinase (p38 MAPK). To identify downstream mediators of the 8-Cl-cAMP signaling, we performed co-immunoprecipitation combined with mass spectrometry using the anti-AMPK or p38 MAPK antibodies. Through this approach, SHC1 was identified as one of the binding partners of p38 MAPK. SHC1 phosphorylation was suppressed by 8-Cl-cAMP in HeLa and MCF7 cancer cells, which was mediated by its metabolites, 8-Cl-adenosine and 8-Cl-ATP; however, 8-Cl-cAMP showed no effect on SHC1 phosphorylation in normal human fibroblasts. SHC1 siRNA induced AMPK and p38 MAPK phosphorylation and growth inhibition in cancer cells, and SHC1 overexpression re-sensitized human foreskin fibroblasts to the 8-Cl-cAMP treatment. SHC1 phosphorylation was unaffected by Compound C (an AMPK inhibitor) and SB203580 (a p38 MAPK inhibitor), which suggests that SHC1 is upstream of AMPK and p38 MAPK in the 8-Cl-cAMP-stimulated signaling cascade. On the basis of these findings, we conclude that SHC1 functions as a sensor during the 8-Cl-cAMP-induced growth inhibition in SHC1-overexpressing cancer cells.

  14. cAMP-mediated stabilization of fusion pores in cultured rat pituitary lactotrophs

    PubMed Central

    Calejo, Ana Isabel; Jorgačevski, Jernej; Kucka, Marek; Kreft, Marko; Gonçalves, Paula Polónia; Stojilkovic, Stanko S.; Zorec, Robert

    2013-01-01

    Regulated exocytosis mediates the release of hormones and transmitters. The last step of this process is represented by the merger between the vesicle and the plasma membranes, and the formation of a fusion pore. Once formed, the initially stable and narrow fusion pore may reversibly widen (transient exocytosis) or fully open (full-fusion exocytosis). Exocytosis is typically triggered by an elevation in cytosolic calcium activity. However, other second messengers, such as cyclic AMP (cAMP), have been reported to modulate secretion. The way in which cAMP influences the transitions between different fusion pore states remains unclear. Here, hormone release studies show that prolactin release from isolated rat lactotrophs stimulated by forskolin, an activator of adenylyl cyclases, and by membrane-permeable cAMP analog (dbcAMP), exhibit a biphasic concentration dependency. While at lower concentrations (2–10 μM forskolin and 2.5–5 mM dbcAMP) these agents stimulate prolactin release, an inhibition is measured at higher concentrations (50 μM forskolin and 10–15 mM dbcAMP). By using high-resolution capacitance (Cm) measurements, we recorded discrete increases in Cm, which represent elementary exocytic events. An elevation of cAMP leaves the frequency of full-fusion events unchanged, while increasing the frequency of transient events. These exhibited a wider fusion pore as measured by increased fusion pore conductance and a prolonged fusion pore dwell-time. The probability of observing rhythmic reopening of transient fusion pores was elevated by dbcAMP. In conclusion, cAMP-mediated stabilization of wide fusion pores prevents vesicles from proceeding to the full-fusion stage of exocytosis, which hinders vesicle content discharge at high cAMP concentrations. PMID:23637196

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

    PubMed Central

    Paramonov, Valeriy M.; Mamaeva, Veronika; Sahlgren, Cecilia; Rivero-Müller, Adolfo

    2015-01-01

    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. PMID:26441653

  16. Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

    PubMed Central

    Zagotta, William N.; Stoll, Stefan

    2014-01-01

    Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron–electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels. PMID:24958877

  17. Isolation and characterization of cAMP-resistant mutants of the H-4 rat hepatoma cells

    SciTech Connect

    Liu, A.Y.; Lin, Z.

    1987-05-01

    H-4 rat hepatoma cells were mutagenized with ethyl methane-sulfonate and the frequency of emergence of cAMP resistant mutant cells were evaluated by cloning the EMS-treated cells in a semi-solid agar medium that contained either 1-3 mM 8-bromo-cAMP plus 1 mM 3-isobutyl-1-methyl xanthine or 5 ..mu..g/ml cholera toxin plus 1 mM IBMX. cAMP resistant mutants emerged at a frequency of 8 x 10/sup -5/. 15 colonies were isolated, recloned, grown in mass culture, and cell extracts were prepared. Analysis of cAMP-dependent protein kinase demonstrated that: (1) the type II enzyme is the only cAMP-dependent protein kinase detected in extracts of the hepatoma cells; (2) of the 15 cAMP resistant clonal cell lines examined, only one (H/sub 4/M/sub 18/) was found to be devoid of cAMP-dependent protein kinase activity. In another cell line (H/sub 4/M/sub 10/) the activity was 30% of that of the parental H-4 cells; (3) there was an increase (130-300%) in cAMP-dependent protein kinase activity in 13/15 of the mutant cell lines over that of the parental H-4 cells. Analysis of cAMP-phosphodiesterase demonstrated significant increases (150-370%) in the enzyme activity in extracts of the mutants over that of the H-4 parental line. Their results suggest that while a deficiency in cAMP-dependent protein kinase may confer resistance to the hepatoma cells against the cytostatic effects of 8-bromo-cAMP and cholera toxin, other events such as overexpression of phosphodiesterase may contribute to this phenotype.

  18. Atmospheric, Magnetospheric and Plasmas in Space (AMPS) spacelab payload definition study. Volume 3: Interface control documents. Part 1: AMPS payload to shuttle ICD

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Physical, functional, and operational interfaces between the space shuttle orbiter and the AMPS payload are described for the ground handling and test phases, prelaunch, launch and ascent, operational, stowage, and reentry and landing activities.

  19. Quantitative Measurement of cAMP Concentration Using an Exchange Protein Directly Activated by a cAMP-Based FRET-Sensor

    PubMed Central

    Salonikidis, Petrus S.; Zeug, André; Kobe, Fritz; Ponimaskin, Evgeni; Richter, Diethelm W.

    2008-01-01

    Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores. Here, we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an “exchange protein directly activated by cAMP”. In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by [cAMP] changes was determined as ΔE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in the range from 150 nM to 15 μM. The method described is also suitable for other FRET-based biosensors with a 1:1 donor/acceptor stoichiometry. As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7). PMID:18708470

  20. cAMP-independent signal pathways stimulate hyphal morphogenesis in Candida albicans.

    PubMed

    Parrino, Salvatore M; Si, Haoyu; Naseem, Shamoon; Groudan, Kevin; Gardin, Justin; Konopka, James B

    2017-03-01

    The fungal pathogen Candida albicans can transition from budding to hyphal growth, which promotes biofilm formation and invasive growth into tissues. Stimulation of adenylyl cyclase to form cAMP induces hyphal morphogenesis. The failure of cells lacking adenylyl cyclase (cyr1Δ) to form hyphae has suggested that cAMP signaling is essential for hyphal growth. However, cyr1Δ mutants also grow slowly and have defects in morphogenesis, making it unclear whether hyphal inducers must stimulate cAMP, or if normal basal levels of cAMP are required to maintain cellular health needed for hyphal growth. Interestingly, supplementation of cyr1Δ cells with low levels of cAMP enabled them to form hyphae in response to the inducer N-acetylglucosamine (GlcNAc), suggesting that a basal level of cAMP is sufficient for stimulation. Furthermore, we isolated faster-growing cyr1Δ pseudorevertant strains that can be induced to form hyphae even though they lack cAMP. The pseudorevertant strains were not induced by CO2 , consistent with reports that CO2 directly stimulates adenylyl cyclase. Mutational analysis showed that induction of hyphae in a pseudorevertant strain was independent of RAS1, but was dependent on the EFG1 transcription factor that acts downstream of protein kinase A. Thus, cAMP-independent signals contribute to the induction of hyphal responses.

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

    ERIC Educational Resources Information Center

    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…

  2. AKAP-mediated feedback control of cAMP gradients in developing hippocampal neurons.

    PubMed

    Gorshkov, Kirill; Mehta, Sohum; Ramamurthy, Santosh; Ronnett, Gabriele V; Zhou, Feng-Quan; Zhang, Jin

    2017-04-01

    Cyclic AMP (cAMP) and protein kinase A (PKA), classical examples of spatially compartmentalized signaling molecules, are critical axon determinants that regulate neuronal polarity and axon formation, yet little is known about micro-compartmentalization of cAMP and PKA signaling and its role in developing neurons. Here, we revealed that cAMP forms a gradient in developing hippocampal neurons, with higher cAMP levels in more distal regions of the axon compared to other regions of the cell. Interestingly, this cAMP gradient changed according to the developmental stage and depended on proper anchoring of PKA by A-kinase anchoring proteins (AKAPs). Disrupting PKA anchoring to AKAPs increased the cAMP gradient in early-stage neurons and led to enhanced axon elongation. Our results provide new evidence for a local negative-feedback loop, assembled by AKAPs, for the precise control of a growth-stage-dependent cAMP gradient to ensure proper axon growth.

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    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. A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

    PubMed Central

    Abdou, Houssein S.; Bergeron, Francis

    2014-01-01

    Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production. PMID:25225331

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

    NASA Astrophysics Data System (ADS)

    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

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

    SciTech Connect

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying

    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

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Cell-to-cell coordination for the spontaneous cAMP oscillation in Dictyostelium

    NASA Astrophysics Data System (ADS)

    Nagano, Seido; Sakurai, Shunsuke

    2013-12-01

    We propose a new cellular dynamics scheme for the spontaneous cAMP oscillations in Dictyostelium discoideum. Our scheme seamlessly integrates both receptor dynamics and G-protein dynamics into our previously developed cellular dynamics scheme. Extensive computer simulation studies based on our new cellular dynamics scheme were conducted in mutant cells to evaluate the molecular network. The validity of our proposed molecular network as well as the controversial PKA-dependent negative feedback mechanism was supported by our simulation studies. Spontaneous cAMP oscillations were not observed in a single mutant cell. However, multicellular states of various mutant cells consistently initiated spontaneous cAMP oscillations. Therefore, cell-to-cell coordination via the cAMP receptor is essential for the robust initiation of spontaneous cAMP oscillations.

  9. In vivo, cAMP stimulates growth and morphogenesis of mouse mammary ducts.

    PubMed

    Silberstein, G B; Strickland, P; Trumpbour, V; Coleman, S; Daniel, C W

    1984-08-01

    In culture, cAMP is known to be mitogenic for mammary cells and several other epithelia, but evidence for a similar role in vivo has been only correlative. We have used plastic implants to cause slow release of cholera toxin and other cAMP-active agents to local areas of mammary glands in ovariectomized mice. Elevated levels of intracellular cAMP around the implants promoted vigorous growth and normal ductal morphogenesis, while distant sites were unaffected. Local effects of cAMP included restoration of normal ductal caliber, formation of new end buds, and reinitiation of DNA synthesis in both epithelium and surrounding stroma. Thus, cAMP is both a mitogenic and a morphogenetic factor in this tissue.

  10. AMPed Up immunity: how antimicrobial peptides have multiple roles in immune defense

    PubMed Central

    Lai, Yuping; Gallo, Richard L.

    2009-01-01

    Antimicrobial peptides (AMPs) are widely expressed and rapidly induced at epithelial surfaces to repel assault from diverse infectious agents including bacteria, viruses, fungi and parasites. Much information suggests that AMPs act by mechanisms that extend beyond their capacity to serve as gene-encoded antibiotics. For example, some AMPs alter the properties of the mammalian membrane or interact with its receptors to influence diverse cellular processes including cytokine release, chemotaxis, antigen presentation, angiogenesis and wound healing. These functions complement their antimicrobial action and favor resolution of infection and repair of damaged epithelia. Opposing this, some microbes have evolved mechanisms to inactivate or avoid AMPs and subsequently become pathogens. Thus, AMPs are multifunctional molecules that have a central role in infection and Inflammation. PMID:19217824

  11. Cyclic AMP Mimics the Anti-ageing Effects of Calorie Restriction by Up-Regulating Sirtuin.

    PubMed

    Wang, Zhuoran; Zhang, Lu; Liang, Yaru; Zhang, Chi; Xu, Zhiyu; Zhang, Lang; Fuji, Ryosuke; Mu, Wei; Li, Liyuan; Jiang, Junjun; Ju, Yong; Wang, Zhao

    2015-07-08

    Cyclic adenosine monophosphate (cAMP) plays an important role in many biological processes as a second messenger, and cAMP treatment has been reported to extend the lifespan of wild-type Drosophila melanogaster. Our study showed that exogenous cAMP improved ageing-related phenotypes by increasing the protein level of Sirtuins, which prevented metabolic disorders to mimic the effect of calorie restriction. Experiments in vitro showed that cAMP directly bound to SIRT1 and SIRT3 and consequently increased their activity. These findings suggest that cAMP slows the ageing process and is a good candidate to mimic calorie restriction. Our research provides a promising therapeutic strategy to target metabolic disorder-induced ageing-related diseases.

  12. Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

    SciTech Connect

    Kangasniemi, M.; Kaipia, A.; Toppari, J.; Mali, P.; Huhtaniemi, I.; Parvinen, M. )

    1990-05-01

    Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis.

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

    PubMed

    Jiang, Jing; Fan, Ling Wen; Wu, Wei Hua

    2005-08-01

    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 Verticillium-secreted toxins by regulating the production of the known signal SA in plant defense pathway.

  14. Dibutyryl cAMP effects on thromboxane and leukotriene production in decompression-induced lung injury

    NASA Technical Reports Server (NTRS)

    Little, T. M.; Butler, B. D.

    1997-01-01

    Decompression-induced venous bubble formation has been linked to increased neutrophil counts, endothelial cell injury, release of vasoactive eicosanoids, and increased vascular membrane permeability. These actions may account for inflammatory responses and edema formation. Increasing the intracellular cAMP has been shown to decrease eicosanoid production and edema formation in various models of lung injury. Reduction of decompression-induced inflammatory responses was evaluated in decompressed rats pretreated with saline (controls) or dibutyryl cAMP (DBcAMP, an analog of cAMP). After pretreatment, rats were exposed to either 616 kPa for 120 min or 683 kPa for 60 min. The observed increases in extravascular lung water ratios (pulmonary edema), bronchoalveolar lavage, and pleural protein in the saline control group (683 kPa) were not evident with DBcAMP treatment. DBcAMP pretreatment effects were also seen with the white blood cell counts and the percent of neutrophils in the bronchoalveolar lavage. Urinary levels of thromboxane B2, 11-dehydrothromboxane B2, and leukotriene E4 were significantly increased with the 683 kPa saline control decompression exposure. DBcAMP reduced the decompression-induced leukotriene E4 production in the urine. Plasma levels of thromboxane B2, 11-dehydrothromboxane B2, and leukotriene E4 were increased with the 683-kPa exposure groups. DBcAMP treatment did not affect these changes. The 11-dehydrothromboxane B2 and leukotriene E4 levels in the bronchoalveolar lavage were increased with the 683 kPa exposure and were reduced with the DBcAMP treatment. Our results indicate that DBcAMP has the capability to reduce eicosanoid production and limit membrane permeability and subsequent edema formation in rats experiencing decompression sickness.

  15. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

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

    PubMed

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R

    2012-06-22

    Tumor necrosis factor α (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 that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF+ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.

  17. Aip regulates cAMP signalling and GH secretion in GH3 cells.

    PubMed

    Formosa, R; Xuereb-Anastasi, A; Vassallo, J

    2013-08-01

    Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been linked to predisposition to pituitary adenomas. However, the mechanism by which this occurs remains unknown. AIP interacts with a number of interesting proteins, including members of the cAMP signalling pathway that has been shown to be consistently altered in pituitary tumours. The functional role of Aip was investigated using both over-expression and knock down of Aip in GH3 cells. cAMP signalling and its downstream effectors, including GH secretion, were then investigated. cAMP signalling was analysed using cAMP assays, cAMP-response element-promoter luciferase reporter assays, real-time PCR and finally secreted GH quantification. Over-expression of wild-type (WT)-Aip reduced forskolin-induced cAMP signalling at the total cAMP level, luciferase reporter activity and target gene expression, when compared with empty vector and the non-functional R304X mutant. Additionally, GH secretion was reduced in WT-Aip over-expressing GH3 cells treated with forskolin. Knock down of endogenous Aip resulted in increased cAMP signalling but a decrease in GH secretion was also noted. Inhibition of phosphodiesterase activity using general and selective inhibitors did not completely ablate the effect of Aip on forskolin-augmented cAMP signalling. A mechanism by which Aip acts as a tumour suppressor, by maintaining a low cAMP signalling and concentration, is suggested. Mutations of Aip render the protein incapable of such activity. This effect appears not to be mediated by the AIP-PDE interaction, suggesting the involvement of other interacting partners in mediating this outcome.

  18. Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex.

    PubMed

    Guérin, François; Isnard, Christophe; Cattoir, Vincent; Giard, Jean Christophe

    2015-12-01

    Enterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-β-D-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

  19. Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

    PubMed Central

    Guérin, François; Isnard, Christophe; Giard, Jean Christophe

    2015-01-01

    Enterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist β-lactams mainly by producing the AmpC β-lactamase enzyme. AmpC expression is highly inducible in the presence of some β-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished β-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-β-d-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level β-lactam resistance, suggesting a post-transcriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level β-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of β-lactam resistance in ECC, which is essential for the identification of novel potential drug targets. PMID:26438498

  20. PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production.

    PubMed

    García-Morales, Verónica; Cuíñas, Andrea; Elíes, Jacobo; Campos-Toimil, Manuel

    2014-03-01

    Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.

  1. cap alpha. /sub 2/-Adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production

    SciTech Connect

    Jones, S.B.; Toews, M.L.; Turner, J.T.; Bylund, D.B.

    1987-03-01

    Preincubation of HT29 human colonic adenocarcinoma cells with ..cap alpha../sub 2/-adrenergic agonists resulted in a 10- to 20-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated without agonist. Similar results were obtained using either a (/sup 3/H)adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, which is termed sensitization, is ..cap alpha../sub 2/-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an ..cap alpha../sub 2/-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, whereas prazosin (..cap alpha../sub 1/-adrenergic) and sotalol (..beta..-adrenergic) did not. The time for half-maximal sensitization was 5 min and the half-time for reversal was 10 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide was observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation with a phosphodiesterase inhibitor and forskolin did not mimic sensitization. Increasing the levels of cyclic AMP during the preincubation had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the growing list of ..cap alpha../sub 2/-adrenergic responses that are not mediated by a decrease in cyclic AMP.

  2. Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

    PubMed Central

    Gausdal, G; Wergeland, A; Skavland, J; Nguyen, E; Pendino, F; Rouhee, N; McCormack, E; Herfindal, L; Kleppe, R; Havemann, U; Schwede, F; Bruserud, Ø; Gjertsen, B T; Lanotte, M; Ségal-Bendirdjian, E; Døskeland, S O

    2013-01-01

    We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients. PMID:23449452

  3. Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis.

    PubMed

    Gausdal, G; Wergeland, A; Skavland, J; Nguyen, E; Pendino, F; Rouhee, N; McCormack, E; Herfindal, L; Kleppe, R; Havemann, U; Schwede, F; Bruserud, O; Gjertsen, B T; Lanotte, M; Ségal-Bendirdjian, E; Døskeland, S O

    2013-02-28

    We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.

  4. Stimulation of limb cartilage differentiation by cyclic AMP is dependent on cell density.

    PubMed

    Rodgers, B J; Kulyk, W M; Kosher, R A

    1989-12-01

    Cyclic AMP (cAMP) has been implicated in the regulation of limb cartilage differentiation. This study represents an attempt to clarify potential mechanisms by which cAMP might regulate chondrogenesis. We have found that the ability of cAMP to stimulate limb cartilage differentiation in vitro is dependent on cell density. Dibutyryl cAMP (dbcAMP) elicits a striking increase in the accumulation of Alcian blue, pH 1.0-positive cartilage matrix, and a corresponding three- to fourfold increase in the accumulation of 35S-labeled glycosaminoglycans (GAG) by limb mesenchymal cells cultured in low serum medium at densities greater than confluence (i.e. micromass cultures established with 1-2 x 10(5) cells in 10 microliters of medium). Moreover, dbcAMP causes a striking (two- to fourfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific sulfated proteoglycan in these high density, supraconfluent cultures. In contrast, cAMP does not promote the chondrogenesis of limb mesenchymal cells cultured at subconfluent densities (i.e. cultures initiated with 2.5-5 x 10(4) cells in 10 microliters of medium). In these low density cultures, dbcAMP does not promote the formation of cartilage matrix, sulfated GAG accumulation or the accumulation of cartilage-specific mRNAs. These observations suggest that cAMP may exert its regulatory effect in part by facilitating cell-cell communication during the critical condensation phase of chondrogenesis.

  5. Perspectives of the AMP-activated kinase (AMPK) signalling pathway in thyroid cancer.

    PubMed

    Andrade, Bruno Moulin; de Carvalho, Denise Pires

    2014-04-01

    Approximately 90% of non-medullary thyroid malignancies originate from the follicular cell and are classified as papillary or follicular (well-differentiated) thyroid carcinomas, showing an overall favourable prognosis. However, recurrence or persistence of the disease occurs in some cases associated with the presence of loco-regional or distant metastatic lesions that generally become resistant to radioiodine therapy, while glucose uptake and metabolism are increased. Recent advances in the field of tumor progression have shown that CTC (circulating tumour cells) are metabolic and genetically heterogeneous. There is now special interest in unravelling the mechanisms that allow the reminiscence of dormant tumour lesions that might be related to late disease progression and increased risk of recurrence. AMPK (AMP-activated protein kinase) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism that are fundamental for cell survival in a stressful environment; nevertheless, the activation of this kinase also decreases cell proliferation rate and induces tumour cell apoptosis. In the thyroid field, AMPK emerged as a novel important intracellular pathway, since it regulates both iodide and glucose uptakes in normal thyroid cells. Furthermore, it has recently been demonstrated that the AMPK pathway is highly activated in papillary thyroid carcinomas, although the clinical significance of these findings remains elusive. Herein we review the current knowledge about the role of AMPK activation in thyroid physiology and pathophysiology, with special focus on thyroid cancer.

  6. Perspectives of the AMP-activated kinase (AMPK) signalling pathway in thyroid cancer

    PubMed Central

    Andrade, Bruno Moulin; de Carvalho, Denise Pires

    2014-01-01

    Approximately 90% of non-medullary thyroid malignancies originate from the follicular cell and are classified as papillary or follicular (well-differentiated) thyroid carcinomas, showing an overall favourable prognosis. However, recurrence or persistence of the disease occurs in some cases associated with the presence of loco-regional or distant metastatic lesions that generally become resistant to radioiodine therapy, while glucose uptake and metabolism are increased. Recent advances in the field of tumor progression have shown that CTC (circulating tumour cells) are metabolic and genetically heterogeneous. There is now special interest in unravelling the mechanisms that allow the reminiscence of dormant tumour lesions that might be related to late disease progression and increased risk of recurrence. AMPK (AMP-activated protein kinase) is activated by the depletion in cellular energy levels and allows adaptive changes in cell metabolism that are fundamental for cell survival in a stressful environment; nevertheless, the activation of this kinase also decreases cell proliferation rate and induces tumour cell apoptosis. In the thyroid field, AMPK emerged as a novel important intracellular pathway, since it regulates both iodide and glucose uptakes in normal thyroid cells. Furthermore, it has recently been demonstrated that the AMPK pathway is highly activated in papillary thyroid carcinomas, although the clinical significance of these findings remains elusive. Herein we review the current knowledge about the role of AMPK activation in thyroid physiology and pathophysiology, with special focus on thyroid cancer. PMID:27919039

  7. Next-step-targeted experiments on the Mega-Amp Spherical Tokamak

    NASA Astrophysics Data System (ADS)

    Gryaznevich, M.; Akers, R. J.; Counsell, G. F.; Cunningham, G.; Dnestrovskij, A.; Field, A. R.; Hender, T. C.; Kirk, A.; Lloyd, B.; Meyer, H.; Morris, A. W.; Sykes, A.; Tabasso, A.; Valovic, M.; Voss, G. M.; Wilson, H. R.

    2003-05-01

    Since its first physics campaign, the principal parameters on MAST (Mega-Amp Spherical Tokamak) [A. Sykes et al., Nuclear Fusion 41, 1423 (2001)] have been brought up towards their design values. Considerable advances have been made in a range of physics areas of direct relevance to the International Thermonuclear Experimental Reactor (ITER) [ITER Physics Basis, Nuclear Fusion 39, 2175 (1999)]. In this paper, results on H-mode access, global confinement and pedestal studies are presented and compared with conventional aspect ratio tokamak scalings. Physics and engineering requirements relevant to next step spherical tokamak devices are discussed, in particular the plasma formation, current ramp-up and sustainment, and plasma exhaust. Results of first experiments directly targeting these issues are presented: Plasma current up to 0.5 MA has been produced without use of the central solenoid flux, and current ramp-up and sustainment without use of the central solenoid flux has been demonstrated. Experiments on neutral beam heating and current drive (CD) demonstrate up to 50% bootstrap current fraction and good CD efficiency, and divertor power loading has been found to be tolerable and have a favorable outboard asymmetry.

  8. cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

    SciTech Connect

    Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui; Ha, Hyunil Lee, Zang Hee

    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.

  9. [Adrenergic beta-2 receptors and cyclic AMP in lymphocytes and their relationship to uterine contractility].

    PubMed

    von Mandach, U

    1994-01-01

    It is especially in the long-term application where the pharmacodynamics of the betamimetics determine their effectiveness. According to the time and dosis, there is a decrease in the density and function of the beta 2-adrenoceptors (desensitization). Clinically, this means a loss of effectiveness. This study investigated whether in the course of a normal pregnancy (n = 22) there is a change in the effectiveness of the betamimetics, as expressed by a change in the number of beta 2-adrenoceptors or their function. The results show a 50% decrease in the number of beta 2-adrenoceptors to the 36th gestational week and an increase to initial values after delivery. A similar pattern is found for the function of the beta 2-adrenoceptors (cyclic AMP). The implications for the uterus might be that, with advancing pregnancy, it becomes less prone to relaxation and that the betaadrenergic system, as a mechanism supporting prepare the way for delivery at term, becomes less significant. For tocolysis with betamimetics, the decrease of the beta 2-adrenoceptor density means that, with increasing gestational age, the responsiveness of the uterus to betamimetics decreases.

  10. AMP-deaminase from thymus of patients with myasthenia gravis.

    PubMed

    Rybakowska, I; Szydłowska, M; Szrok, S; Bakuła, S; Kaletha, K

    2015-01-01

    Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes.

  11. Functional modulation of AMP-activated protein kinase by cereblon.

    PubMed

    Lee, Kwang Min; Jo, Sooyeon; Kim, Hyunyoung; Lee, Jongwon; Park, Chul-Seung

    2011-03-01

    Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

  12. Polynomial solutions of the Monge-Ampère equation

    SciTech Connect

    Aminov, Yu A

    2014-11-30

    The question of the existence of polynomial solutions to the Monge-Ampère equation z{sub xx}z{sub yy}−z{sub xy}{sup 2}=f(x,y) is considered in the case when f(x,y) is a polynomial. It is proved that if f is a polynomial of the second degree, which is positive for all values of its arguments and has a positive squared part, then no polynomial solution exists. On the other hand, a solution which is not polynomial but is analytic in the whole of the x, y-plane is produced. Necessary and sufficient conditions for the existence of polynomial solutions of degree up to 4 are found and methods for the construction of such solutions are indicated. An approximation theorem is proved. Bibliography: 10 titles.

  13. AMP-activated protein kinase and metabolic control

    PubMed Central

    Viollet, Benoit; Andreelli, Fabrizio

    2011-01-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. It is now recognized that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile and blood pressure in insulin-resistant rodents. Indeed, AMPK activation mimics the beneficial effects of physical activity or those of calorie restriction by acting on multiple cellular targets. In addition it is now demonstrated that AMPK is one of the probable (albeit indirect) targets of major antidiabetic drugs including, the biguanides (metformin) and thiazolidinediones, as well as of insulin sensitizing adipokines (e.g., adiponectin). Taken together, such findings highlight the logic underlying the concept of targeting the AMPK pathway for the treatment of metabolic syndrome and type 2 diabetes. PMID:21484577

  14. Activation of Exchange Protein Activated by Cyclic-AMP Enhances Long-Lasting Synaptic Potentiation in the Hippocampus

    ERIC Educational Resources Information Center

    Gelinas, Jennifer N.; Banko, Jessica L.; Peters, Melinda M.; Klann, Eric; Weeber, Edwin J.; Nguyen, Peter V.

    2008-01-01

    cAMP is a critical second messenger implicated in synaptic plasticity and memory in the mammalian brain. Substantial evidence links increases in intracellular cAMP to activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream effectors (transcription factors, receptors, protein kinases) necessary for long-term…

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

    SciTech Connect

    Kucka, Marek; Pogrmic-Majkic, Kristina; Fa, Svetlana; Stojilkovic, Stanko S.; Kovacevic, Radmila

    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

  16. Fibrotic lung fibroblasts show blunted inhibition by cAMP due to deficient cAMP response element-binding protein phosphorylation.

    PubMed

    Liu, Xiaoqiu; Sun, Shu Qiang; Ostrom, Rennolds S

    2005-11-01

    Pulmonary fibroblasts regulate extracellular matrix production and degradation; thus, they are critical for maintenance of lung structure, function, and repair. In pulmonary fibrosis, fibroblasts produce excess collagen and form fibrotic foci that eventually impair lung function, but the mechanisms responsible for these alterations are not known. Receptors coupled to the stimulation of cAMP production can inhibit activation of fibroblasts and thereby are antifibrotic. To test whether this signaling pathway is altered in pulmonary fibrosis, we compared the ability of normal adult human pulmonary fibroblasts to generate and respond to cAMP with that of cells isolated from lungs with idiopathic pulmonary fibrosis. Serum- and transforming growth factor (TGF)-beta-stimulated cell proliferation was inhibited approximately 50% by forskolin and approximately 100% by prostaglandin (PG) E(2) in the normal cells but substantially less in the diseased cells. Collagen synthesis was also inhibited >50% by the same drugs in the normal cells but significantly less so in the diseased cells, despite responding with similar increases in cAMP production. Although expression of protein kinase A (PKA) and cAMP-stimulated PKA activity were similar in both the normal and diseased cell types, forskolin- and PGE(2)-stimulated cAMP response element-binding protein (CREB) phosphorylation was decreased in the diseased cell lines compared with the normal cells. cAMP-mediated activation and TGF-beta-mediated inhibition of CREB DNA binding was also diminished in the diseased cells. Thus, pulmonary fibroblasts derived from patients with pulmonary fibrosis are refractory to the inhibition by cAMP due to altered activity of components distal to the activity of PKA, in particular the phosphorylation of CREB.

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

    PubMed Central

    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

  18. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

  19. Linking cellular actin status with cAMP signaling in Candida albicans.

    PubMed

    Wang, Yue; Zou, Hao; Fang, Hao-Ming; Zhu, Yong

    2010-01-01

    The fungal pathogen Candida albicans has a remarkable ability to switch growth forms. Particularly, the yeast-to-hyphae switch is closely linked with its virulence. A range of chemicals and conditions can promote hyphal growth including serum, peptidoglycan, CO2, neutral pH, and elevated temperature. All these signals act essentially through the adenylyl cyclase Cyr1 that synthesizes cAMP. Cells lacking Cyr1 are completely defective in hyphal growth. Recently, cellular actin status is found to influence cAMP synthesis. However, how Cyr1 senses and processes multiple external and internal signals to produce a contextually proper level of cAMP remains unclear. We hypothesized that Cyr1 itself possesses multiple sensors for different signals and achieves signal integration through a combined allosteric effect on the catalytic center. To test this hypothesis, we affinity-purified a Cyr1-containing complex and found that it could enhance cAMP synthesis upon treatment with serum, peptidoglycan or CO2 in vitro. The data indicate that the complex is an essentially intact sensor/effector apparatus for cAMP synthesis. The complex contains two more subunits, the cyclase-associated protein Cap1 and G-actin. We discovered that G-actin plays a regulatory role, rendering cAMP synthesis responsive to actin dynamics. These findings shed new lights on the mechanisms that regulate cAMP-mediated responses in fungi.

  20. Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA

    PubMed Central

    Jones, Christopher P; Ferré-D'Amaré, Adrian R

    2014-01-01

    Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression. PMID:25271255

  1. Evidence for cAMP as a mediator of gonadotropin secretion from male pituitaries

    SciTech Connect

    Bourne, G.A.; Baldwin, D.M.

    1987-09-01

    The purpose of this study was to use sodium flufenamate, a compound that inhibits gonadotropin-releasing hormone (GnRH)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in the pituitary, to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion from male pituitaries. Quartered male pituitaries were perifused at 37/sup 0/C and sequential effluent fractions collected every 10 min. Infusions of GnRH resulted in a twofold increase in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Cycloheximide, 5 ..mu..M, completely inhibited the GnRH-stimulated LH and FSH secretion. Infusions of 0.1 mM flufenamate had similar effects on gonadotropin secretion as cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate restored the secretory responses of both hormones. The flufenamate-inhibited GnRH stimulated LH and FSH release, which was restored by DBcAMP and appeared to be protein synthesis dependent and specific for cAMP.These results suggest an indirect role for cAMP as a mediator of gonadotropin secretion from male pituitaries. However, in contrast to female pituitaries, the secretion of these hormones form male pituitaries is completely dependent on cAMP and de novo protein synthesis.

  2. Spatiotemporal Coupling of cAMP Transporter to CFTR Chloride Channel Function in the Gut Epithelia

    PubMed Central

    Li, Chunying; Krishnamurthy, Partha C.; Penmatsa, Himabindu; Marrs, Kevin L.; Wang, Xue Qing; Zaccolo, Manuela; Jalink, Kees; Li, Min; Nelson, Deborah J.; Schuetz, John D.; Naren, Anjaparavanda P.

    2007-01-01

    SUMMARY Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, is functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. MRP4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea. PMID:18045536

  3. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation

    PubMed Central

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to 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 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 in Teff cells 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. PMID:27621729

  4. Spatiotemporal coupling of cAMP transporter to CFTR chloride channel function in the gut epithelia.

    PubMed

    Li, Chunying; Krishnamurthy, Partha C; Penmatsa, Himabindu; Marrs, Kevin L; Wang, Xue Qing; Zaccolo, Manuela; Jalink, Kees; Li, Min; Nelson, Deborah J; Schuetz, John D; Naren, Anjaparavanda P

    2007-11-30

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel localized at apical cell membranes and exists in macromolecular complexes with a variety of signaling and transporter molecules. Here, we report that the multidrug resistance protein 4 (MRP4), a cAMP transporter, functionally and physically associates with CFTR. Adenosine-stimulated CFTR-mediated chloride currents are potentiated by MRP4 inhibition, and this potentiation is directly coupled to attenuated cAMP efflux through the apical cAMP transporter. CFTR single-channel recordings and FRET-based intracellular cAMP dynamics suggest that a compartmentalized coupling of cAMP transporter and CFTR occurs via the PDZ scaffolding protein, PDZK1, forming a macromolecular complex at apical surfaces of gut epithelia. Disrupting this complex abrogates the functional coupling of cAMP transporter activity to CFTR function. Mrp4 knockout mice are more prone to CFTR-mediated secretory diarrhea. Our findings have important implications for disorders such as inflammatory bowel disease and secretory diarrhea.

  5. Effects of selective phosphodiesterase inhibition on cyclic AMP hydrolysis in rat cerebral cortical slices.

    PubMed Central

    Challiss, R. A.; Nicholson, C. D.

    1990-01-01

    1. The effects of selective inhibition of phosphodiesterase activities on the concentration and rate of hydrolysis of adenosine 3':5' cyclic-monophosphate (cyclic AMP) in rat cerebral cortical slices has been studied. 2. Isoprenaline caused a rapid, concentration-dependent increase in cyclic AMP concentration to new steady-state levels (basal: 7.1 +/- 0.7; 10 microM isoprenaline: 14.3 +/- 1.4 pmol mg-1 protein). Addition of a beta-adrenoceptor antagonist to isoprenaline-stimulated cerebral cortical slices caused a rapid decrease in cyclic AMP concentration to basal levels (t1/2: 58 +/- 18 s). 3. Preincubation of slices for 30 min with the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine, denbufylline, rolipram or Ro20,1724 caused concentration-dependent increases in basal and isoprenaline-stimulated cyclic AMP concentrations and decreased the rate of cyclic AMP hydrolysis measured after addition of a beta-adrenoceptor antagonist. However, SKF 94120 and zaprinast had none of these effects. 4. The results are discussed with respect to previous studies of phosphodiesterase isozymic activities isolated from cerebrum and it is suggested that the Ca2+/calmodulin-independent, low Km cyclic AMP phosphodiesterase isozyme, which is selectively inhibited by denbufylline, rolipram and Ro20,1724, and is present in cerebrum is of critical importance to the regulation of cyclic AMP concentration in this tissue. PMID:2158837

  6. Cyclic-AMP regulation of calcium-dependent K channels in an insect central neurone.

    PubMed

    David, J A; Pitman, R M

    1996-01-26

    In the cockroach fast coxal depressor motoneurone, either the muscarinic agonist McN-A-343 or dibutyryl cAMP (Db-cAMP) induced a reduction in voltage-dependent outward current. The response to McN is due to suppression of a calcium-dependent potassium current (IK,Ca) produced secondarily to a reduction in voltage-dependent calcium current (ICa). The response to Db-cAMP was investigated in order to establish whether cAMP might mediate the response to McN. ICa was suppressed by 3-isobutyl-1-methylxanthine (IBMX) but not by Db-cAMP. The effects of IBMX were therefore unlikely to be the result of phosphodiesterase inhibition. Since caffeine also suppressed ICa, the observed effect of IBMX is probably due to release of Ca2+ from intracellular stores. IK,Ca, evoked by injection of Ca2+, was reduced by Db-cAMP or forskolin but not by McN. These results indicate that the electrical response to McN in this neurone is not mediated by changes in cAMP.

  7. Multi-physics modelling contributions to investigate the atmospheric cosmic rays on the single event upset sensitivity along the scaling trend of CMOS technologies.

    PubMed

    Hubert, G; Regis, D; Cheminet, A; Gatti, M; Lacoste, V

    2014-10-01

    Particles originating from primary cosmic radiation, which hit the Earth's atmosphere give rise to a complex field of secondary particles. These particles include neutrons, protons, muons, pions, etc. Since the 1980s it has been known that terrestrial cosmic rays can penetrate the natural shielding of buildings, equipment and circuit package and induce soft errors in integrated circuits. Recently, research has shown that commercial static random access memories are now so small and sufficiently sensitive that single event upsets (SEUs) may be induced from the electronic stopping of a proton. With continued advancements in process size, this downward trend in sensitivity is expected to continue. Then, muon soft errors have been predicted for nano-electronics. This paper describes the effects in the specific cases such as neutron-, proton- and muon-induced SEU observed in complementary metal-oxide semiconductor. The results will allow investigating the technology node sensitivity along the scaling trend.

  8. High Precision Thermal, Structural and Optical Analysis of an External Occulter Using a Common Model and the General Purpose Multi-Physics Analysis Tool Cielo

    NASA Technical Reports Server (NTRS)

    Hoff, Claus; Cady, Eric; Chainyk, Mike; Kissil, Andrew; Levine, Marie; Moore, Greg

    2011-01-01

    The efficient simulation of multidisciplinary thermo-opto-mechanical effects in precision deployable systems has for years been limited by numerical toolsets that do not necessarily share the same finite element basis, level of mesh discretization, data formats, or compute platforms. Cielo, a general purpose integrated modeling tool funded by the Jet Propulsion Laboratory and the Exoplanet Exploration Program, addresses shortcomings in the current state of the art via features that enable the use of a single, common model for thermal, structural and optical aberration analysis, producing results of greater accuracy, without the need for results interpolation or mapping. This paper will highlight some of these advances, and will demonstrate them within the context of detailed external occulter analyses, focusing on in-plane deformations of the petal edges for both steady-state and transient conditions, with subsequent optical performance metrics including intensity distributions at the pupil and image plane.

  9. Operational, hyper-resolution hydrologic modeling over the contiguous U.S. using themulti-scale, multi-physics WRF-Hydro Modeling and Data Assimilation System.

    NASA Astrophysics Data System (ADS)

    Gochis, D. J.; Cosgrove, B.; Yu, W.; Clark, E. P.; Yates, D. N.; Dugger, A. L.; McCreight, J. L.; Pan, L.; Zhang, Y.; rafeei-Nasab, A.; Karsten, L. R.; Cline, D. W.; Sampson, K. M.; Newman, A. J.; Wood, A.; Win-Gildenmeister, M.

    2015-12-01

    Operational flood, flash flood and water supply forecasting is typically conducted using a host of different observational and modeling tools that range widely in process complexity, spatial resolution andobservational data sources. While such tailored approaches can provide significant skill in specific water forecasting applications, the lack of a more coordinated general approach can result in inconsistency between various forecast products and can inhibit transfer of information, methodologies between forecast systems. With the aim of improving the timeliness, consistency and spatial fidelity hydrologic prediction products, the U.S. National Weather Service has initiated an effort to provide street-level, water prediction services for the nation. This effort seeks to incorporate advances in hydrometeorological observing capabilities, new hydrologic data assimilation methodologies, improvements in hydrographic and geospatial information and advances in the ulitizion of high performance computers for process-based hydrologic modeling. This talk will summarize the proposed Initial Operating Capability (IOC) for national water prediction using the community WRF-Hydro modeling system, scheduled for operational execution during late spring of 2016. Four different configurations of the WRF-Hydro system are planned including an Analysis and Data Assimilation configuration, Short Range (0-2 day) and Medium Range (0-10 day) deterministic configurations and a Long Range (0-30 day) enesmble configuration. Streamflow analyses and forecasts from each model configurations will be produced on 2.7 million river reaches of the NHDPlusv2 hydrographic dataset. This presentation summarizes results from a number of different model development and benchmarking activities conducted as part of the IOC effort. Results from prototype real-time forecasting activities conducted during the 2015 National Flood Interoperability Experiment (NFIE) will be presented as will retrospective

  10. Activation of f-channels by cAMP analogues in macropatches from rabbit sino-atrial node myocytes.

    PubMed Central

    Bois, P; Renaudon, B; Baruscotti, M; Lenfant, J; DiFrancesco, D

    1997-01-01

    1. The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp-cAMPs and Sp-cAMPs, was investigated on hyperpolarization-activated 'pacemaker' current (i(f)) recorded in inside-out macropatches from rabbit sino-atrial (SA) node myocytes. 2. When superfused on the intracellular side of f-channels at the concentration of 10 microM, both cAMP derivatives accelerated i(f) activation; their action was moderately less pronounced than that due to the same concentration of cAMP. 3. The measurement of the i(f) conductance-voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of i(f) to more positive voltages with no change in maximal (fully activated) conductance. 4. Dose-response relationships of the shift of the i(f) activation curve showed that both Rp-cAMPs and Sp-cAMPs act as agonists in the cAMP-dependent direct f-channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 microM, and Hill coefficients of 0.82 and 1.12 for Sp-cAMPs and Rp-cAMPs, respectively. 5. The activating action of Rp-cAMPs, a known antagonist of cAMP in the activation of cAMP-dependent protein kinase, confirms previously established evidence that f-channel activation does not involve phosphorylation. These results also suggest that the cAMP binding site of f-channels may be structurally similar to the cyclic nucleotide binding site of olfactory receptor channels. PMID:9218217

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

    NASA Astrophysics Data System (ADS)

    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.

  12. A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death

    PubMed Central

    Wang, Z; Liu, D; Varin, A; Nicolas, V; Courilleau, D; Mateo, P; Caubere, C; Rouet, P; Gomez, A-M; Vandecasteele, G; Fischmeister, R; Brenner, C

    2016-01-01

    Although cardiac cytosolic cyclic 3′,5′-adenosine monophosphate (cAMP) regulates multiple processes, such as beating, contractility, metabolism and apoptosis, little is known yet on the role of this second messenger within cardiac mitochondria. Using cellular and subcellular approaches, we demonstrate here the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1), and show a protective role for sACt against cell death, apoptosis as well as necrosis in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3−) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increases the mitochondrial membrane potential (ΔΨm) and ATP production. cAMP is rate limiting for matrix Ca2+ entry via Epac1 and the mitochondrial calcium uniporter and, as a consequence, prevents mitochondrial permeability transition (MPT). The mitochondrial cAMP effects involve neither protein kinase A, Epac2 nor the mitochondrial Na+/Ca2+ exchanger. In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3− rescued the sensitization of mitochondria to Ca2+-induced MPT. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism and cell death in the heart, which is independent of cytosolic cAMP signaling. Our results might have implications for therapeutic prevention of cell death in cardiac pathologies. PMID:27100892

  13. Intraocular injection of dibutyryl cyclic AMP promotes axon regeneration in rat optic nerve.

    PubMed

    Monsul, Nicholas T; Geisendorfer, Abram R; Han, Paul J; Banik, Rudrani; Pease, Mary Ellen; Skolasky, Richard L; Hoffman, Paul N

    2004-04-01

    The optic nerve is a CNS pathway containing molecules capable of inhibiting axon elongation. The growth program in embryonic retinal ganglion cell (RGC) neurons enables axons to regenerate in the optic nerve through at least two mechanisms. Namely, high cyclic AMP (cAMP) levels abrogate the ability of CNS molecules to inhibit elongation, and the pattern of gene expression enables axons to undergo rapid, sustained, and lengthy elongation. In adult mammals, recovery of visual function after optic nerve injury is limited by both the death of most RGC neurons and the inability of surviving axons to regenerate. We now report that a single intraocular injection of the membrane-permeable cAMP analogue dibutyryl cAMP (db cAMP) promotes the regeneration of RGC axons in the optic nerves of adult rats, but does not prevent the death of RGC neurons. This regeneration in optic nerves crushed within the orbit (2 mm from the eye) was equally effective either 1 day before or 1 day after db cAMP injection. The number of regenerating axons, which was maximal 14 days after crush, declined with increasing time after injury (i.e., 28, 56, and 112 days) and distance beyond the crush site (i.e., 0.25, 0.5, and 1.0 mm). Thus, db cAMP promotes optic nerve regeneration without increasing the survival of axotomized RGC neurons. Furthermore, since db cAMP does not enable axons to undergo rapid, sustained, and lengthy elongation, strategies that increase survival and promote these changes in elongation may critically complement the ability of db cAMP to promote regeneration.

  14. Role of cyclic AMP in pulmonary xenobiotic metabolism with special emphasis on benzo(a)pyrene

    SciTech Connect

    Schaeffer, V.H.

    1986-01-01

    This thesis was intended to investigate the role of the intracellular regulator, cAMP, on pulmonary xenobiotic metabolism using the well-studied carcinogen, benzo(a)pyrene (BP) as a representative xenobiotic. Lung slices from rats administered N/sup 6/, O/sup 2/', dibutyryl cAMP (DcAMP), theophylline or forskolin, all of which elevated biologically reactive cAMP levels in the lung, showed an increased ability to metabolize (/sup 3/H)-BP. This effect occurred beyond 6 hr following treatment and reached a maximum at 12 hr, at a time when cAMP content had already peaked and returned to basal levels. The perfusion of BP through the isolated lungs of animals administered DcAMP in vivo indicated that the BP metabolites primarily responsible for the cyclic nucleotide-induced increase in metabolism were the 3-hydroxy BP, 9-hydroxy BP, BP 9, 10 diol, BP-glucuronides and BP-glutathione conjugates. Kinetic analysis indicated that the Km component of these reactions was altered without a corresponding change in Vmax, suggesting that elevated pulmonary cAMP content may be affecting the detoxication enzymes, UDP-glucuronyltransferase and sulfotransferase. Studies with pulmonary microsomes from DcAMP-treated animals indicated that the cyclic nucleotide not only enhanced the hydroxylation of BP but also the cytochrome P450-dependent hydroxylation of coumarin. This is supported by the fact that DcAMP administration in vivo also enhanced phosphorylation of two classes of nuclear proteins, histones and nuclear acidic proteins, believed to play a role in the transcription of RNA and DNA.

  15. Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

    SciTech Connect

    Shearer, W.T.; Patke, C.L.; Gilliam, E.B.; Rosenblatt, H.M.; Barron, K.S.; Orson, F.M.

    1988-09-01

    A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

  16. Cyclic AMP-dependent protein kinase regulates basal and cyclic AMP-stimulated but not phorbol ester-stimulated transcription of the tyrosine hydroxylase gene.

    PubMed

    Kim, K S; Tinti, C; Song, B; Cubells, J F; Joh, T H

    1994-09-01

    To define the precise role of cyclic AMP (cAMP)-dependent protein kinase (PKA) in transcriptional regulation of the tyrosine hydroxylase (TH) gene, we performed transient cotransfection analyses of a reporter construct containing the upstream 2,400 bp sequence of the rat TH gene with expression plasmids encoding a heat-stable specific inhibitor of PKA (PKI), a mutant regulatory subunit of PKA, or the catalytic subunit of PKA. Inhibition of PKA activity by expression of either PKI or mutant regulatory subunit blocked cAMP-stimulated induction and reduced basal transcription of the TH-reporter construct. Expression of the catalytic subunit of PKA induced the expression of the TH-reporter construct up to 50-fold in a dose-dependent manner. Primer extension analysis confirmed that PKA-mediated induction of TH-reporter expression occurred at the correct transcription initiation site. Expression of PKI did not affect induction following phorbol ester treatment, suggesting that PKA and protein kinase C (PKC) induce TH transcription by independent mechanisms. Finally, a double mutation within the cAMP response element (CRE) of TH2400-CAT diminished its basal and forskolin-stimulated transcription to the level of the promoterless plasmid, pBLCAT3, but did not alter the induction following treatment with phorbol ester, indicating that the CRE is not required for PKC-mediated transcriptional induction. Our results indicate that PKA, via the CRE, plays a crucial role for basal and cAMP-inducible transcription of the TH gene.

  17. cAMP analogs and their metabolites enhance TREK-1 mRNA and K+ current expression in adrenocortical cells.

    PubMed

    Enyeart, Judith A; Liu, Haiyan; Enyeart, John J

    2010-03-01

    bTREK-1 K(+) channels set the resting membrane potential of bovine adrenal zona fasciculata (AZF) cells and function pivotally in the physiology of cortisol secretion. Adrenocorticotropic hormone controls the function and expression of bTREK-1 channels through signaling mechanisms that may involve cAMP and downstream effectors including protein kinase A (PKA) and exchange protein 2 directly activated by cAMP (Epac2). Using patch-clamp and Northern blot analysis, we explored the regulation of bTREK-1 mRNA and K(+) current expression by cAMP analogs and several of their putative metabolites in bovine AZF cells. At concentrations sufficient to activate both PKA and Epac2, 8-bromoadenosine-cAMP enhanced the expression of both bTREK-1 mRNA and K(+) current. N(6)-Benzoyladenosine-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K(+) current measured at times from 24 to 96 h. An Epac-selective cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8CPT-2'-OMe-cAMP), potently stimulated bTREK-1 mRNA and K(+) current expression, whereas the nonhydrolyzable Epac activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, Sp-isomer was ineffective. Metabolites of 8CPT-2'-OMe-cAMP, including 8-(4-chlorophenylthio)-2'-O-methyladenosine-5'-O-monophosphate and 8CPT-2'-OMe-adenosine, promoted the expression of bTREK-1 transcripts and ion current with a temporal pattern, potency, and effectiveness resembling that of the parent compound. Likewise, at low concentrations, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 30 microM) but not its nonhydrolyzable analog 8-(4-chlorophenylthio)-cAMP, Sp-isomer, enhanced the expression of bTREK-1 mRNA and current. 8CPT-cAMP metabolites, including 8CPT-adenosine and 8CPT-adenine, also increased bTREK-1 expression. These results indicate that cAMP increases the expression of bTREK-1 mRNA and K(+) current through a cAMP-dependent but Epac2-independent mechanism. They further demonstrate that one or more metabolites of 8

  18. Phase A conceptual design study of the Atmospheric, Magnetospheric and Plasmas in Space (AMPS) payload

    NASA Technical Reports Server (NTRS)

    1974-01-01

    The 12 month Phase A Conceptual Design Study of the Atmospheric, Magnetospheric and Plasmas in Space (AMPS) payload performed within the Program Development Directorate of the Marshall Space Flight Center is presented. The AMPS payload makes use of the Spacelab pressurized module and pallet, is launched by the space shuttle, and will have initial flight durations of 7 days. Scientific instruments including particle accelerators, high power transmitters, optical instruments, and chemical release devices are mounted externally on the Spacelab pallet and are controlled by the experimenters from within the pressurized module. The capability of real-time scientist interaction on-orbit with the experiment is a major characteristic of AMPS.

  19. Compartmentation of cAMP signalling in cardiomyocytes in health and disease.

    PubMed

    Perera, R K; Nikolaev, V O

    2013-04-01

    3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger critically involved in the regulation of heart function. It has been shown to act in discrete subcellular signalling compartments formed by differentially localized receptors, phosphodiesterases and protein kinases. Cardiac diseases such as hypertrophy or heart failure are associated with structural and functional remodelling of these microdomains which leads to changes in cAMP compartmentation. In this review, we will discuss recent key findings which provided new insights into cAMP compartmentation in cardiomyocytes with a particular focus on its alterations in heart disease.

  20. The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases

    PubMed Central

    Raker, Verena Katharina; Becker, Christian; Steinbrink, Kerstin

    2016-01-01

    Nucleotide signaling molecules contribute to the regulation of cellular pathways. In the immune system, cyclic adenosine monophosphate (cAMP) is well established as a potent regulator of innate and adaptive immune cell functions. Therapeutic strategies to interrupt or enhance cAMP generation or effects have immunoregulatory potential in autoimmune and inflammatory disorders. Here, we provide an overview of the cyclic AMP axis and its role as a regulator of immune functions and discuss the clinical and translational relevance of interventions with these processes. PMID:27065076

  1. Strategies Targeting cAMP Signaling in the Treatment of Polycystic Kidney Disease

    PubMed Central

    Harris, Peter C.

    2014-01-01

    Polycystic kidney disease (PKD) is a leading cause of ESRD worldwide. In PKD, excessive cell proliferation and fluid secretion, pathogenic interactions of mutated epithelial cells with an abnormal extracellular matrix and alternatively activated interstitial macrophages, and the disruption of mechanisms controlling tubular diameter contribute to cyst formation. Studies with animal models suggest that several diverse pathophysiologic mechanisms, including dysregulation of intracellular calcium levels and cAMP signaling, mediate these cystogenic mechanisms. This article reviews the evidence implicating calcium and cAMP as central players in a network of signaling pathways underlying the pathogenesis of PKD and considers the therapeutic relevance of treatment strategies targeting cAMP signaling. PMID:24335972

  2. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-15

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening.

  3. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed Central

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-01

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening. PMID:1846445

  4. HeLa human cervical cancer cell migration is inhibited by treatment with dibutyryl-cAMP.

    PubMed

    Lee, Jae-Wook; Lee, Jiyoung; Moon, Eun-Yi

    2014-07-01

    Cyclic AMP (cAMP) activates both protein kinase A (PKA) and guanine-nucleotide exchange factor exchange protein directly activated by CAMP (EPAC)-mediated Ras-related Protein1 (RAP1) GTPase that regulates various cellular functions including cell migration. Herein, we investigated whether cAMP-mediated PKA and EPAC1/RAP1 pathways differentially control HeLa cervical cancer cell migration. Although HeLa cell migration was reduced by dibutyryl-cAMP, we observed an increase in cAMP/PKA, cAMP/EPAC1/RAP1-GTPase, and RAC1-GTPase. HeLa cell migration and RAC1-GTPase were increased by treatment with 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP analogue to activate EPAC-specific signaling pathways. When HeLa cells were treated with H-89, a PKA inhibitor, cell migration was enhanced but RAC1-GTPase was inhibited. In addition, cell migration induced by dibutyryl-cAMP was reversed but the activity of Rac1-GTPase was inhibited by H-89 treatment. Taken together, these data demonstrate that cAMP/PKA and cAMP/EPAC1/RAP1-GTPase might inversely control cervical cancer cell migration, although both signaling pathways may up-regulate RAC1-GTPase. It also suggests that cAMP-mediated cancer cell migration was independent of RAC1-GTPase activation.

  5. Removal characteristics of CO2 using aqueous MEA/AMP solutions in the absorption and regeneration process.

    PubMed

    Choi, Won-Joon; Seo, Jong-Beom; Jang, Sang-Yong; Jung, Jong-Hyeon; Oh, Kwang-Joong

    2009-01-01

    The carbon dioxide (CO2) removal efficiency, reaction rate, and CO2 loading into aqueous blended monoethanolamine (MEA) + 2-amino-2-methyl-1-propanol (AMP) solutions to enhance absorption characteristics of MEA and AMP were carried out by the absorption/regeneration process. As a result, compared to aqueous MEA and AMP solutions, aqueous blended MEA + AMP solutions have a higher CO2 loading than MEA and a higher reaction rate than AMP. The CO2 loading of rich amine of aqueous 18 wt.% MEA + 12 wt.% AMP solution was 0.62 mol CO2/mol amine, which is 51.2% more than 30 wt.% MEA (0.41 mol CO2/mol amine). Consequently, blending MEA and AMP could be an effective way to design considering economical efficiency and used to operate absorber for a long time.

  6. Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum.

    PubMed

    Hara, Shuichi; Kobayashi, Masamune; Kuriiwa, Fumi; Mukai, Toshiji; Mizukami, Hajime

    2012-03-15

    Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of

  7. Antitumor Trans Platinum Adducts of GMP and AMP

    PubMed Central

    Liu, Yangzhong; Sivo, Maria F.; Natile, Giovanni

    2000-01-01

    Recently it has been shown that several analogues of the clinically ineffective trans-DDP exhibit antitumor activity comparable to that of cis-DDP. The present paper describes the binding of antitumor trans-[PtCl2(E-iminoether)2] (trans-EE) to guanosinemonophosphate (GMP) and adenosinemonophosphate (AMP). We have used HPLC and 1H and 15N NMR to characterize the different adducts. In the case of a 1:1 mixture of trans-EE and GMP, at an early stage of the reaction, a monofunctional adduct is formed which, subsequently, is partly converted into a monosolvated monofunctional species. After about 70 hours an equilibrium is established between chloro and solvato monofunctional adducts at a ratio of 30/70. In the presence of excess GMP (4:1) the initially formed monofunctional adducts react further to give two bifunctional adducts, one with the iminoether ligands in their original E configurations and the other with the iminoether ligands having one E and the other, Z configurations. The coordination geometry obtained by energy minimization calculations is in qualitative agreement with 2D NMR data. PMID:18475942

  8. Effects of AMP-activated protein kinase in cerebral ischemia.

    PubMed

    Li, Jun; McCullough, Louise D

    2010-03-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to 'cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed.

  9. Effects of AMP-activated protein kinase in cerebral ischemia

    PubMed Central

    Li, Jun; McCullough, Louise D

    2010-01-01

    AMP-activated protein kinase (AMPK) is a serine threonine kinase that is highly conserved through evolution. AMPK is found in most mammalian tissues including the brain. As a key metabolic and stress sensor/effector, AMPK is activated under conditions of nutrient deprivation, vigorous exercise, or heat shock. However, it is becoming increasingly recognized that changes in AMPK activation not only signal unmet metabolic needs, but also are involved in sensing and responding to ‘cell stress', including ischemia. The downstream effect of AMPK activation is dependent on many factors, including the severity of the stressor as well as the tissue examined. This review discusses recent in vitro and in vivo studies performed in the brain/neuronal cells and vasculature that have contributed to our understanding of AMPK in stroke. Recent data on the potential role of AMPK in angiogenesis and neurogenesis and the interaction of AMPK with 3-hydroxy-3-methy-glutaryl-CoA reductase inhibitors (statins) agents are highlighted. The interaction between AMPK and nitric oxide signaling is also discussed. PMID:20010958

  10. Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis.

    PubMed

    Takiar, Vinita; Nishio, Saori; Seo-Mayer, Patricia; King, J Darwin; Li, Hui; Zhang, Li; Karihaloo, Anil; Hallows, Kenneth R; Somlo, Stefan; Caplan, Michael J

    2011-02-08

    Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves both fluid secretion and abnormal proliferation of cyst-lining epithelial cells. The chloride channel of the cystic fibrosis transmembrane conductance regulator (CFTR) participates in secretion of cyst fluid, and the mammalian target of rapamycin (mTOR) pathway may drive proliferation of cyst epithelial cells. CFTR and mTOR are both negatively regulated by AMP-activated protein kinase (AMPK). Metformin, a drug in wide clinical use, is a pharmacological activator of AMPK. We find that metformin stimulates AMPK, resulting in inhibition of both CFTR and the mTOR pathways. Metformin induces significant arrest of cystic growth in both in vitro and ex vivo models of renal cystogenesis. In addition, metformin administration produces a significant decrease in the cystic index in two mouse models of ADPKD. Our results suggest a possible role for AMPK activation in slowing renal cystogenesis as well as the potential for therapeutic application of metformin in the context of ADPKD.

  11. Automated image analysis of FRET signals for subcellular cAMP quantification.

    PubMed

    Leavesley, Silas J; Nakhmani, Arie; Gao, Yi; Rich, Thomas C

    2015-01-01

    A variety of FRET probes have been developed to examine cAMP localization and dynamics in single cells. These probes offer a readily accessible approach to measure localized cAMP signals. However, given the low signal-to-noise ratio of most FRET probes and the dynamic nature of the intracellular environment, there have been marked limitations in the ability to use FRET probes to study localized signaling events within the same cell. Here, we outline a methodology to dissect kinetics of cAMP-mediated FRET signals in single cells using automated image analysis approaches. We additionally extend these approaches to the analysis of subcellular regions. These approaches offer an unique opportunity to assess localized cAMP kinetics in an unbiased, quantitative fashion.

  12. [Vibrational spectra of monoclinic diphosphates of formula AMP2O7].

    PubMed

    Serghini Idrissi, M; Rghioui, L; Nejjar, R; Benarafa, L; Saidi Idrissi, M; Lorriaux, A; Wallart, F

    2004-07-01

    The monoclinic pyrophosphates with AMP2O7 formula were synthesized. Their infrared and Raman spectra have been reported and analysed. The results of a force field calculation for CaCuP2O7 are presented.

  13. N-Acetyl-D- and L-esters of 5'-AMP hydrolyze at different rates

    NASA Technical Reports Server (NTRS)

    Wickramasinghe, N. S.; Lacey, J. C. Jr; Lacey JC, J. r. (Principal Investigator)

    1993-01-01

    Studies of the properties of aminoacyl derivatives of 5'-AMP are aimed at understanding the origin of the process of protein synthesis. Aminoacyl (2',3') esters of 5'-AMP can serve as models of the 3'-terminus of aminoacyl tRNA. We report here on the relative rates of hydrolysis of Ac-D- and L-Phe AMP esters as a function of pH. At all pHs above 3, the rate constant of hydrolysis of the Ac-L-Phe ester is 1.7 to 2.1 times that of Ac-D-Phe ester. The D-isomer seems partially protected from hydrolysis by a stronger association with the adenine ring of the 5'-AMP.

  14. Atmospheric, Magnetospheric, and Plasmas in Space (AMPS) spacelab payload definition study, appendixes

    NASA Technical Reports Server (NTRS)

    Keeley, J. T.

    1976-01-01

    An equipment list, instrument baseline data, engineering drawings, mass properties computer printouts, electrical energy management, and control and display functional analysis pertinent to the AMPS (Satellite Payload) are presented.

  15. Use of phenylthiocarbamide for assessing cAMP-dependent resistance to anoxia in animals.

    PubMed

    Bolekhan, E A; Semenov, D G; Gerasimova, I A; Samoilov, M O

    1997-01-01

    The responses of cats with different levels of taste sensitivity to phenylthiocarbamide (PTC) bitters to five-minute hypoxia were studied; PTC sensitivity is a genetic marker of the activity of the cAMP system. Animals able to perceive PTC showed a number of functional differences, with higher levels of resistance to anoxia, than those which could not perceive PTC. The groups showed significant differences in the basal cAMP content in the cerebral cortex, and in the time course of changes in the cAMP level during anoxia and subsequent reoxygenation. It is suggested that these differences result from genetically determined features of the cAMP system, which is involved in forming adaptive responses.

  16. A QM/MM study of the 5‧-AMP DNA hydrolysis of aprataxin

    NASA Astrophysics Data System (ADS)

    Hanaoka, Kyohei; Tanaka, Wataru; Kayanuma, Megumi; Shoji, Mitsuo

    2015-07-01

    Aprataxin is a DNA repair enzyme that hydrolyzes the abnormal 5‧-AMP termini of broken DNAs. Based on quantum mechanical/molecular mechanical (QM/MM) calculations, we found that the catalytic reaction proceeds in three steps; substrate protonation, DNA deadenylation and histidine-AMP intermediate hydrolysis. The calculated activation energies for the second and third reactions are 19.0 and 10.5 kcal mol-1, which can be attributed to a penta-coordinated AMP-phosphoryl formation and closing of a water molecule, respectively. We also found that a histidine-AMP intermediate is hydrolyzed easily in the third step when a water molecule closes within 3 Å to the phosphorus nucleus.

  17. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    SciTech Connect

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki . E-mail: yk-kim@korea.ac.kr

    2007-05-18

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway.

  18. Atmospheric, Magnetospheric, and Plasmas in Space (AMPS) spacelab payload definition study, technical summary document

    NASA Technical Reports Server (NTRS)

    Keeley, J. T.

    1976-01-01

    Some 60 instrument candidates and 80 possible science investigations were evaluated. The early analysis emphasized the science aspect in terms of the functional requirements for each of the potential experiments identified by the AMPS science working group. These requirements were then used for the grouping of instruments into practical payloads which would fit the capabilities of the Shuttle/Spacelab. This analysis resulted in the definition of eleven different AMPS configurations. The data were then used to define a typical set of requirements for a flexible AMPS laboratory. The data gathered to this point showed that a planned sequential buildup of the laboratory would be necessary to meet both physical and funding limitations. This led to the definition of five strawman payloads by the science working group, which were used to establish a conceptual laboratory and to define preliminary design of a configuration which could satisfy AMPS needs during the early program period.

  19. Big defensins and mytimacins, new AMP families of the Mediterranean mussel Mytilus galloprovincialis.

    PubMed

    Gerdol, Marco; De Moro, Gianluca; Manfrin, Chiara; Venier, Paola; Pallavicini, Alberto

    2012-02-01

    Antimicrobial peptides (AMPs) play a fundamental role in the innate immunity of invertebrates, preventing the invasion of potential pathogens. Mussels can express a surprising abundance of cysteine-rich AMPs pertaining to the defensin, myticin, mytilin and mytimycin families, particularly in the circulating hemocytes. Based on deep RNA sequencing of Mytilus galloprovincialis, we describe the identification, molecular diversity and constitutive expression in different tissues of five novel transcripts pertaining to the macin family (named mytimacins) and eight novel transcripts pertaining to the big defensins family (named MgBDs). The predicted antimicrobial peptides exhibit a N-terminal signal peptide, a positive net charge and a high content in cysteines, allegedly organized in intra-molecular disulfide bridges. Mytimacins and big defensins therefore represent two novel AMP families of M. galloprovincialis which extend the repertoire of cysteine-rich AMPs in this bivalve mollusk.

  20. A forskolin derivative, colforsin daropate hydrochloride, inhibits rat mesangial cell mitogenesis via the cyclic AMP pathway.

    PubMed

    Ogata, Junichi; Minami, Kouichiro; Segawa, Kayoko; Yamamoto, Chieko; Kim, Sung-Teh; Shigematsu, Akio

    2003-11-01

    A forskolin derivative, colforsin daropate hydrochloride (CDH), has been introduced as an inotropic agent that acts directly on adenylate cyclase to increase intracellular cyclic AMP (cAMP) levels and ventricular contractility, resulting in positive inotropic activity. We investigated the effects of CDH on rat mesangial cell (MC) proliferation. CDH (10(-7)-10(-5) mol/l) inhibited [(3)H]thymidine incorporation into cultured rat MCs in a concentration-dependent manner. CDH (10(-7)-10(-5) mol/l) also decreased cell numbers in a similar manner, and stimulated cAMP accumulation in MCs in a concentration-dependent manner. A protein kinase A inhibitor, H-89, abolished the inhibitory effects of CDH on MC mitogenesis. These findings suggest that CDH would inhibit the proliferation of rat MCs via the cAMP pathway.

  1. Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes

    SciTech Connect

    Gelerstein, S.; Shapira, H.; Dascal, N.; Yekuel, R.; Oron, Y.

    1988-05-01

    Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of /sup 45/Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

    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.

  3. Measurement of cAMP in an undergraduate teaching laboratory, using ALPHAscreen technology.

    PubMed

    Bartho, Joseph D; Ly, Kien; Hay, Debbie L

    2012-02-14

    Adenosine 3',5'-monophosphate (cAMP) is a cellular second messenger with central relevance to pharmacology, cell biology, and biochemistry teaching programs. cAMP is produced from adenosine triphosphate by adenylate cyclase, and its production is reduced or enhanced upon activation of many G protein-coupled receptors. Therefore, the measurement of cAMP serves as an indicator of receptor activity. Although there are many assays available for measuring cAMP, few are suitable for large class teaching, and even fewer seem to have been adapted for this purpose. Here, we describe the use of bead-based ALPHAscreen (Amplified Luminescent Proximity Homogenous Assay) technology for teaching a class of more than 300 students the practical aspects of detecting signal transduction. This technology is applicable to the measurement of many different signaling pathways. This resource is designed to provide a practical guide for instructors and a useful model for developing other classes using similar technologies.

  4. Quercetin promotes neurite growth through enhancing intracellular cAMP level and GAP-43 expression.

    PubMed

    Chen, Ming-Ming; Yin, Zhi-Qi; Zhang, Lu-Yong; Liao, Hong

    2015-09-01

    The present study was designed to investigate the role of quercetin on neurite growth in N1E-115 cells and the underlying mechanisms. Quercetin was evaluated for its effects on cell numbers of neurites, neurite length, intracellular cAMP content, and Gap-43 expression in N1E-115 cells in vitro by use of microscopy, LANCE(tm) cAMP 384 kit, and Western blot analysis, respectively. Our results showed that quercetin could increase the neurite length in a concentration-dependent manner, but had no effect on the numbers of cells. Quercetin significantly increased the expression of cellular cAMP in a time- and concentration-dependent manner. The Gap-43 expression was up-regulated in a time-dependent manner. In conclusion, quercetin could promote neurite growth through increasing the intracellular cAMP level and Gap-43 expression.

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

    PubMed

    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

  6. Vv-AMP1, a ripening induced peptide from Vitis vinifera shows strong antifungal activity

    PubMed Central

    de Beer, Abré; Vivier, Melané A

    2008-01-01

    Background Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors. Results A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal

  7. Structural mechanisms in the abolishment of VEGF-induced microvascular hyperpermeability by cAMP.

    PubMed

    Fu, Bingmei M; Shen, Shang; Chen, Bin

    2006-06-01

    To investigate the structural mechanisms by which elevation of the intraendothelial cAMP levels abolishes or attenuates the transient increase in microvascular permeability by vascular endothelial growth factor (VEGF), we examined cAMP effect on VEGF-induced hyperpermeability to small solute sodium fluorescein (Stokes radius = 0.45 nm) P(sodium fluorescein), intermediate-sized solute alpha-lactalbumin (Stokes radius = 2.01 nm) P(alpha-lactalbumin), and large solute albumin (BSA, Stokes radius = 3.5 nm) P(BSA) on individually perfused microvessels of frog mesenteries. After 20 min pretreatment of 2 mM cAMP analog, 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P(sodium fluorescein) (from a peak increase of 2.6+/-0.37 times control with VEGF alone to 0.96+/-0.07 times control with VEGF and cAMP), in P(alpha-lactalbumin) (from a peak increase of 2.7+/-0.33 times control with VEGF alone to 0.76+/-0.07 times control with VEGF and cAMP), and in P(BSA) (from a peak increase of 6.5+/-1.0 times control with VEGF alone to 0.97+/-0.08 times control with VEGF and cAMP). Based on these measured data, the prediction from our mathematical models suggested that the increase in the number of tight junction strands in the cleft between endothelial cells forming the microvessel wall is one of the mechanisms for the abolishment of VEGF-induced hyperpermeability by cAMP.

  8. Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet β-Cells

    PubMed Central

    Dezaki, Katsuya; Damdindorj, Boldbaatar; Sone, Hideyuki; Dyachok, Oleg; Tengholm, Anders; Gylfe, Erik; Kurashina, Tomoyuki; Yoshida, Masashi; Kakei, Masafumi; Yada, Toshihiko

    2011-01-01

    OBJECTIVE Ghrelin reportedly restricts insulin release in islet β-cells via the Gαi2 subtype of G-proteins and thereby regulates glucose homeostasis. This study explored whether ghrelin regulates cAMP signaling and whether this regulation induces insulinostatic cascade in islet β-cells. RESEARCH DESIGN AND METHODS Insulin release was measured in rat perfused pancreas and isolated islets and cAMP production in isolated islets. Cytosolic cAMP concentrations ([cAMP]i) were monitored in mouse MIN6 cells using evanescent-wave fluorescence imaging. In rat single β-cells, cytosolic protein kinase-A activity ([PKA]i) and Ca2+ concentration ([Ca2+]i) were measured by DR-II and fura-2 microfluorometry, respectively, and whole cell currents by patch-clamp technique. RESULTS Ghrelin suppressed glucose (8.3 mmol/L)-induced insulin release in rat perfused pancreas and isolated islets, and these effects of ghrelin were blunted in the presence of cAMP analogs or adenylate cyclase inhibitor. Glucose-induced cAMP production in isolated islets was attenuated by ghrelin and enhanced by ghrelin receptor antagonist and anti-ghrelin antiserum, which counteract endogenous islet-derived ghrelin. Ghrelin inhibited the glucose-induced [cAMP]i elevation and [PKA]i activation in MIN6 and rat β-cells, respectively. Furthermore, ghrelin potentiated voltage-dependent K+ (Kv) channel currents without altering Ca2+ channel currents and attenuated glucose-induced [Ca2+]i increases in rat β-cells in a PKA-dependent manner. CONCLUSIONS Ghrelin directly interacts with islet β-cells to attenuate glucose-induced cAMP production and PKA activation, which lead to activation of Kv channels and suppression of glucose-induced [Ca2+]i increase and insulin release. PMID:21788571

  9. Cyclic AMP-inducible genes respond uniformly to seasonal lighting conditions in the rat pineal gland.

    PubMed

    Spessert, R; Gupta, B B P; Rohleder, N; Gerhold, S; Engel, L

    2006-12-01

    The encoding of photoperiodic information ensues in terms of the daily profile in the expression of cyclic AMP (cAMP)-inducible genes such as the arylalkylamine N-acetyltransferase (AA-NAT) gene that encodes the rate-limiting enzyme in melatonin formation. In the present study, we compared the influence of the photoperiodic history on the cAMP-inducible genes AA-NAT, inducible cyclic AMP early repressor (ICER), fos-related antigen-2 (FRA-2), mitogen-activated protein kinase phosphatase-1 (MKP-1), nerve growth factor inducible gene-A (NGFI-A) and nerve growth factor inducible gene-B (NGFI-B) in the pineal gland of rats. For this purpose, we monitored the daily profiles of each gene in the same pineal gland under a long (light/dark 16:8) and a short (light/dark 8:16) photoperiod by measuring the respective mRNA amounts by real-time polymerase chain reaction analysis. We found that, for all genes under investigation, the duration of increased nocturnal expression is lengthened and, in relation to light onset, the nocturnal rise is earlier under the long photoperiod (light/dark 16:8). Furthermore, with the exception of ICER, all other cAMP-inducible genes tend to display higher maximum expression under light/dark 8:16 than under light/dark 16:8. Photoperiod-dependent changes persist for all of the cAMP-inducible genes when the rats are kept for two cycles under constant darkness. Therefore, all cAMP-inducible genes are also influenced by the photoperiod of prior entrained cycles. Our study indicates that, despite differences regarding the expressional control and the temporal phasing of the daily profile, cAMP-inducible genes are uniformly influenced by photoperiodic history in the rat pineal gland.

  10. Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

    PubMed Central

    Gard, D L; Lazarides, E

    1982-01-01

    The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation. Images PMID:6294504

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

    NASA Technical Reports Server (NTRS)

    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.

  12. SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP

    PubMed Central

    Shimizu-Albergine, Masami; Van Yserloo, Brian; Golkowski, Martin G.; Ong, Shao-En; Beavo, Joseph A.; Bornfeldt, Karin E.

    2016-01-01

    Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP–SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP

  13. Estradiol increases cAMP in the oviductal secretory cells through a nongenomic mechanism.

    PubMed

    Oróstica, María L; Lopez, John; Rojas, Israel; Rocco, Jocelyn; Díaz, Patricia; Reuquén, Patricia; Cardenas, Hugo; Parada-Bustamante, Alexis; Orihuela, Pedro A

    2014-09-01

    In the rat oviduct, estradiol (E2) accelerates egg transport by a nongenomic action that requires previous conversion of E2 to methoxyestrogens via catechol-O-methyltranferase (COMT) and activation of estrogen receptor (ER) with subsequent production of cAMP and inositol triphosphate (IP3). However, the role of the different oviductal cellular phenotypes on this E2 nongenomic pathway remains undetermined. The aim of this study was to investigate the effect of E2 on the levels of cAMP and IP3 in primary cultures of secretory and smooth muscle cells from rat oviducts and determine the mechanism by which E2 increases cAMP in the secretory cells. In the secretory cells, E2 increased cAMP but not IP3, while in the smooth muscle cells E2 decreased cAMP and increased IP3. Suppression of protein synthesis by actinomycin D did not prevent the E2-induced cAMP increase, but this was blocked by the ER antagonist ICI 182 780 and the inhibitors of COMT OR 486, G protein-α inhibitory (Gαi) protein pertussis toxin and adenylyl cyclase (AC) SQ 22536. Expression of the mRNA for the enzymes that metabolizes estrogens, Comt, Cyp1a1, and Cyp1b1 was found in the secretory cells, but this was not affected by E2. Finally, confocal immunofluorescence analysis showed that E2 induced colocalization between ESR1 (ERα) and Gαi in extranuclear regions of the secretory cells. We conclude that E2 differentially regulates cAMP and IP3 in the secretory and smooth muscle cells of the rat oviduct. In the secretory cells, E2 increases cAMP via a nongenomic action that requires activation of COMT and ER, coupling between ESR1 and Gαi, and stimulation of AC.

  14. Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii.

    PubMed

    Pasquale, S M; Goodenough, U W

    1987-11-01

    When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.

  15. Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

    PubMed

    Shibasaki, T; Takahashi, T; Takahashi, H; Seino, S

    2014-09-01

    Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic β-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²⁺ , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in β-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic β-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes.

  16. Functional roles of arcA, etrA, cyclic AMP (cAMP)-cAMP receptor protein, and cya in the arsenate respiration pathway in Shewanella sp. strain ANA-3.

    PubMed

    Murphy, Julie N; Durbin, K James; Saltikov, Chad W

    2009-02-01

    Microbial arsenate respiration can enhance arsenic release from arsenic-bearing minerals--a process that can cause arsenic contamination of water. In Shewanella sp. strain ANA-3, the arsenate respiration genes (arrAB) are induced under anaerobic conditions with arsenate and arsenite. Here we report how genes that encode anaerobic regulator (arcA and etrA [fnr homolog]) and carbon catabolite repression (crp and cya) proteins affect arsenate respiration in ANA-3. Transcription of arcA, etrA, and crp in ANA-3 was similar in cells grown on arsenate and cells grown under aerobic conditions. ANA-3 strains lacking arcA and etrA showed minor to moderate growth defects, respectively, with arsenate. However, crp was essential for growth on arsenate. In contrast to the wild-type strain, arrA was not induced in the crp mutant in cultures shifted from aerobic to anaerobic conditions containing arsenate. This indicated that cyclic AMP (cAMP)-cyclic AMP receptor (CRP) activates arr operon transcription. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the arr promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase (cya) genes were identified in the genome. One particular cya-like gene was differentially expressed under aerobic versus arsenate respiration conditions. Moreover, a double mutant lacking two of the cya-like genes could not grow with arsenate as a terminal electron acceptor; exogenous cAMP could complement growth of the double cya mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in Shewanella sp. strain ANA-3.

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

    PubMed

    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.

  18. Eviprostat activates cAMP signaling pathway and suppresses bladder smooth muscle cell proliferation.

    PubMed

    Li, Kai; Yao, Jian; Chi, Yuan; Sawada, Norifumi; Araki, Isao; Kitamura, Masanori; Takeda, Masayuki

    2013-06-06

    Eviprostat is a popular phytotherapeutic agent for the treatment of lower urinary tract symptoms (LUTS). At present, the signaling mechanisms underlying its therapeutic effects are still poorly understood. Given that cAMP has been reported to suppress cell hyperplasia and hypertrophy in various pathological situations, we asked whether the effect of Eviprostat could be ascribed to the activation of the cAMP signaling pathway. In the study, exposure of cAMP response element (CRE)-secreted alkaline phosphatase (SEAP) (CRE-SEAP)-reporter cells to Eviprostat elevated SEAP secretion, which was associated with an increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and cAMP-response element-binding protein (CREB), as well as enhanced expression of CRE-regulated protein connexin43, indicating an activation of the cAMP signaling pathway. Consistent with these observations, Eviprostat-induced expression of Cx43 was abolished in the presence of adenylyl cyclase inhibitor SQ22536 or PKA inhibitor H89, whereas it was mimicked by adenylyl cyclase activator, forskolin. Further analysis demonstrated that Eviprostat significantly potentiated the effect of phosphodiesterase 3 (PDE3) inhibitor, but not that of PDE4 inhibitor, on CRE activation. Moreover, Eviprostat suppressed PDGF-induced activation of ERK and Akt and inhibited cell proliferation and hillock formation in both mesangial cells and bladder smooth muscle cells. Collectively, activation of the cAMP signaling pathway could be an important mechanism by which Eviprostat exerts its therapeutic effects for LUTS.

  19. Reactive oxygen species mediate phorbol ester-stimulated cAMP response in human eosinophils.

    PubMed

    Ezeamuzie, Charles I; Taslim, Najla

    2006-08-14

    Recently, we showed that phorbol 12-myristate 13-acetate (PMA) can cause a direct, PKC-dependent, stimulation of intracellular cAMP in human eosinophils. Since PMA also stimulates the release of reactive oxygen species in these cells, we have investigated whether reactive oxygen species are involved in the cAMP response. Provided eosinophils were incubated for <20 min at 37 degrees C before stimulation, PMA potently stimulated cAMP generation that surpassed that of histamine. Pre-treatment of the cells with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI) and apocynin, strongly inhibited the cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O(2)(-)). The cAMP response was also inhibited by pre-treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or NG-nitro-l-arginine methyl ester (L-NAME), thus, suggesting the possible involvement of a peroxide rather than O(2)(-) or nitric oxide (NO). These results reveal a novel involvement of intracellular reactive oxygen species in protein kinase C (PKC)-dependent stimulation of cAMP production in human eosinophils.

  20. The effect of hypoxia on PGE2-stimulated cAMP generation in HMEC-1.

    PubMed

    Wiktorowska-Owczarek, Anna; Owczarek, Jacek

    2015-06-01

    Prostaglandin E2 (PGE2) is generated in various cells, including endothelial cells, and is responsible for various functions, such as vascular relaxation and angiogenesis. Effects of PGE2 are mediated via receptors EP1-EP4, among which EP2 and EP4 are coupled to Gs protein which activates adenylate cyclase (AC) and cAMP synthesis. The aim of this work was to study the ability of human microvascular endothelial cells (HMEC-1) to synthesize cAMP in the presence of PGE2, and to determine the effect of hypoxia on the PGE2- stimulated cAMP level. It was decided to evaluate the effect of PGE2 on the secretion of VEGF, an inducer of angiogenesis. In summary, our findings show that PGE2 induces cAMP production, but hypoxia may impair PGE2-stimulated activity of the AC-cAMP signaling pathway. These results suggest that the cardioprotective effect of PGE2/EP4/cAMP may be attenuated during ischemia. Furthermore, this study indicates that the pro-angiogenic effect of PGE2 is not associated with VEGF secretion in HMEC-1 cells.

  1. Regulation of cAMP Intracellular Levels in Human Platelets Stimulated by 2-Arachidonoylglycerol.

    PubMed

    Signorello, Maria Grazia; Leoncini, Giuliana

    2016-05-01

    We demonstrated that in human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) decreased dose- and time-dependently cAMP intracellular levels. No effect on cAMP decrease induced by 2-AG was observed in the presence of the adenylate cyclase inhibitor SQ22536 as well in platelets pretreated with the thromboxane A2 receptor antagonist, SQ29548 or with aspirin, inhibitor of arachidonic acid metabolism through the cyclooxygenase pathway. An almost complete recovering of cAMP level was measured in platelets pretreated with the specific inhibitor of phosphodiesterase (PDE) 3A, milrinone. In platelets pretreated with LY294002 or MK2206, inhibitors of PI3K/AKT pathway, and with U73122, inhibitor of phospholipase C pathway, only a partial prevention was shown. cAMP intracellular level depends on synthesis by adenylate cyclase and hydrolysis by PDEs. In 2-AG-stimulated platelets adenylate cyclase activity seems to be unchanged. In contrast PDEs appear to be involved. In particular PDE3A was specifically activated, as milrinone reversed cAMP reduction by 2-AG. 2-AG enhanced PDE3A activity through its phosphorylation. The PI3K/AKT pathway and PKC participate to this PDE3A phosphorylation/activation mechanism as it was greatly inhibited by platelet pretreatment with LY294002, MK2206, U73122, or the PKC specific inhibitor GF109203X. Taken together these data suggest that 2-AG potentiates its power of platelet agonist reducing cAMP intracellular level.

  2. Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons.

    PubMed

    Wu, Dongsheng; Zhang, Yi; Bo, Xuenong; Huang, Wenlong; Xiao, Fang; Zhang, Xinyu; Miao, Tizong; Magoulas, Charalambos; Subang, Maria C; Richardson, Peter M

    2007-03-01

    A conditioning lesion to peripheral axons of primary sensory neurons accelerates regeneration of their central axons in vivo or neurite outgrowth if the neurons are grown in vitro. Previous evidence has implicated neuropoietic cytokines and also cyclic AMP in regenerative conditioning. In experiments reported here, delivery through a lentivirus vector of ciliary neurotrophic factor to the appropriate dorsal root ganglion in rats was sufficient to mimic the conditioning effect of peripheral nerve injury on the regeneration of dorsal spinal nerve root axons. Regeneration in this experimental preparation was also stimulated by intraganglionic injection of dibutyryl cyclic AMP but the effects of ciliary neurotrophic factor and dibutyryl cyclic AMP were not additive. Dibutyryl cyclic AMP injection into the dorsal root ganglion induced mRNAs for two other neuropoietic cytokines, interleukin-6 and leukemia inhibitory factor and increased the accumulation of phosphorylated STAT3 in neuronal nuclei. The in vitro conditioning action of dibutyryl cyclic AMP was partially blocked by a pharmacological inhibitor of Janus kinase 2, a neuropoietic cytokine signaling molecule. We suggest that the beneficial actions of increased cyclic AMP activity on axonal regeneration of primary sensory neurons are mediated, at least in part, through the induction of neuropoietic cytokine synthesis within the dorsal root ganglion.

  3. Cyclic AMP relaxes swine arterial smooth muscle predominantly by decreasing cell Ca2+ concentration.

    PubMed Central

    McDaniel, N L; Rembold, C M; Richard, H M; Murphy, R A

    1991-01-01

    1. Our objective was to evaluate the mechanism of cyclic AMP-dependent arterial smooth muscle relaxation. Cyclic AMP-dependent relaxation has been proposed to result from either (a) a decrease in intracellular [Ca2+] or (b) a decrease in [Ca2+] sensitivity of myosin light chain kinase by protein kinase A-dependent phosphorylation of myosin kinase. 2. We evaluated these proposed mechanisms by examining forskolin-induced changes in aequorin-estimated myoplasmic [Ca2+], [cyclic AMP], myosin phosphorylation and stress generation in agonist-stimulated or KCl-depolarized swine common carotid media tissues. 3. Forskolin, an activator of adenylyl cyclase, increased [cyclic AMP] and reduced [Ca2+], myosin phosphorylation and stress in tissues pre-contracted with phenylephrine or histamine. This relaxation was not associated with an alteration of the [Ca2+] sensitivity of phosphorylation, nor the dependence of stress on phosphorylation. 4. Forskolin pre-treatment attenuated, but did not abolish, agonist-induced increases in [Ca2+] and stress. 5. These results suggest that cyclic AMP-induced relaxation of the agonist-stimulated swine carotid media is primarily caused by cyclic AMP-mediated decreases in myoplasmic [Ca2+]. PMID:1654411

  4. Constitutional abnormalities of chromosome 21 predispose to iAMP21-acute lymphoblastic leukaemia.

    PubMed

    Harrison, Christine J; Schwab, Claire

    2016-03-01

    In addition to Down syndrome, individuals with other constitutional abnormalities of chromosome 21 have an increased risk of developing childhood acute lymphoblastic leukaemia (ALL). Specifically, carriers of the Robertsonian translocation between chromosomes 15 and 21, rob(15;21) (q10; q10)c, have ∼2,700 increased risk of developing ALL with iAMP21 (intrachromosomal amplification of chromosome 21). In these patients, chromosome 15 as well as chromosome 21 is involved in the formation of iAMP21, referred to here as der(21)(15;21). Individuals with constitutional ring chromosomes involving chromosome 21, r(21)c, are also predisposed to iAMP21-ALL, involving the same series of mutational processes as seen in sporadic- and der(21)(15;21)-iAMP21 ALL. Evidence is accumulating that the dicentric nature of the Robertsonian and ring chromosome is the initiating factor in the formation of the complex iAMP21 structure. Unravelling these intriguing predispositions to iAMP21-ALL may provide insight into how other complex rearrangements arise in cancer.

  5. The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages

    PubMed Central

    Libanova, Rimma; Lienenklaus, Stefan; Weiss, Siegfried; Guzmán, Carlos A.

    2014-01-01

    The cyclic di-nucleotide bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) is a candidate mucosal adjuvant with proven efficacy in preclinical models. It was shown to promote specific humoral and cellular immune responses following mucosal administration. To date, there is only fragmentary knowledge on the cellular and molecular mode of action of c-di-AMP. Here, we report on the identification of dendritic cells and macrophages as target cells of c-di-AMP. We show that c-di-AMP induces the cell surface up-regulation of T cell co-stimulatory molecules as well as the production of interferon-β. Those responses were characterized by in vitro experiments with murine and human immune cells and in vivo studies in mice. Analyses of dendritic cell subsets revealed conventional dendritic cells as principal responders to stimulation by c-di-AMP. We discuss the impact of the reported antigen presenting cell activation on the previously observed adjuvant effects of c-di-AMP in mouse immunization studies. PMID:24755640

  6. Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion

    PubMed Central

    Gerbaud, Pascale; Taskén, Kjetil; Pidoux, Guillaume

    2015-01-01

    During human placentation, mononuclear cytotrophoblasts fuse to form multinucleated syncytia ensuring hormonal production and nutrient exchanges between the maternal and fetal circulation. Syncytial formation is essential for the maintenance of pregnancy and for fetal growth. The cAMP signaling pathway is the major route to trigger trophoblast fusion and its activation results in phosphorylation of specific intracellular target proteins, in transcription of fusogenic genes and assembly of macromolecular protein complexes constituting the fusogenic machinery at the plasma membrane. Specificity in cAMP signaling is ensured by generation of localized pools of cAMP controlled by cAMP phosphodiesterases (PDEs) and by discrete spatial and temporal activation of protein kinase A (PKA) in supramolecular signaling clusters inside the cell organized by A-kinase-anchoring proteins (AKAPs) and by organization of signal termination by protein phosphatases (PPs). Here we present original observations on the available components of the cAMP signaling pathway in the human placenta including PKA, PDE, and PP isoforms as well as AKAPs. We continue to discuss the current knowledge of the spatiotemporal regulation of cAMP signaling triggering trophoblast fusion. PMID:26441659

  7. Identification of Novel Genes Responsible for Overexpression of ampC in Pseudomonas aeruginosa PAO1

    PubMed Central

    Tsutsumi, Yuko; Tomita, Haruyoshi

    2013-01-01

    The development of resistance to antipseudomonal penicillins and cephalosporins mediated by the chromosomal ampC gene in Pseudomonas aeruginosa is of clinical importance. We isolated piperacillin-resistant mutants derived from P. aeruginosa PAO1 and analyzed two mutants that had an insertion in mpl and nuoN. One mutant, YT1677, was resistant to piperacillin and ceftazidime and had an insertion in mpl, which encodes UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase. The other mutant, YT7988, showed increased MICs of piperacillin, ceftazidime, cefepime, and cefoperazone, and the insertion was mapped to nuoN, which encodes NADH dehydrogenase I chain N. Complementation experiments demonstrated that these mutations resulted in higher levels of resistance to β-lactams. The expression of genes reported to be involved in β-lactam resistance was examined by real-time PCR in YT1677 and YT7988 mutants. Overexpression was observed for only ampC, and other genes were expressed normally. Deletion of the ampR gene in YT1677 and YT7988 resulted in decreased expression of ampC, indicating that the mutations in YT1677 and YT7988 affected the expression of ampC through the function of AmpR. PMID:24041903

  8. Cyclic AMP enhances calcium-dependent potassium current in Aplysia neurons.

    PubMed

    Ewald, D; Eckert, R

    1983-12-01

    The effect on the Ca-dependent potassium current, IK(Ca), of procedures that increase intracellular cAMP levels was studied in Aplysia neurons using three different pharmacological approaches. Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase in IK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP. Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increased IK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied. Application of serotonin plus phosphodiesterase inhibitor caused an increase in IK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease in IK(Ca). It is proposed that elevation of cyclic AMP mediates an increased sensitivity of the IK(Ca) channel to Ca ions.

  9. Role of the energy sensor AMP-activated protein kinase in renal physiology and disease.

    PubMed

    Hallows, Kenneth R; Mount, Peter F; Pastor-Soler, Núria M; Power, David A

    2010-05-01

    The ultrasensitive energy sensor AMP-activated protein kinase (AMPK) orchestrates the regulation of energy-generating and energy-consuming pathways. AMPK is highly expressed in the kidney where it is reported to be involved in a variety of physiological and pathological processes including ion transport, podocyte function, and diabetic renal hypertrophy. Sodium transport is the major energy-consuming process in the kidney, and AMPK has been proposed to contribute to the coupling of ion transport with cellular energy metabolism. Specifically, AMPK has been identified as a regulator of several ion transporters of significance in renal physiology, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), the Na(+)-K(+)-2Cl(-) cotransporter (NKCC), and the vacuolar H(+)-ATPase (V-ATPase). Identified regulators of AMPK in the kidney include dietary salt, diabetes, adiponectin, and ischemia. Activation of AMPK in response to adiponectin is described in podocytes, where it reduces albuminuria, and in tubular cells, where it reduces glycogen accumulation. Reduced AMPK activity in the diabetic kidney is associated with renal accumulation of triglyceride and glycogen and the pathogenesis of diabetic renal hypertrophy. Acute renal ischemia causes a rapid and powerful activation of AMPK, but the functional significance of this observation remains unclear. Despite the recent advances, there remain significant gaps in the present understanding of both the upstream regulating pathways and the downstream substrates for AMPK in the kidney. A more complete understanding of the AMPK pathway in the kidney offers potential for improved therapies for several renal diseases including diabetic nephropathy, polycystic kidney disease, and ischemia-reperfusion injury.

  10. Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment

    PubMed Central

    Godinho, Rosely Oliveira; Costa-Jr, Valter Luiz

    2003-01-01

    This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid. PMID:12642402

  11. The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

    PubMed Central

    Leronni, Daniela

    2013-01-01

    Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures. PMID:23897891

  12. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  13. Different cAMP sources are critically involved in G protein–coupled receptor CRHR1 signaling

    PubMed Central

    dos Santos Claro, Paula A.; Senin, Sergio A.; Maccarrone, Giuseppina; Turck, Christoph W.

    2016-01-01

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein–dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. PMID:27402953

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

    PubMed

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

    2016-07-18

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

  15. Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP.

    PubMed

    Hu, Lei; Santoro, Bina; Saponaro, Andrea; Liu, Haiying; Moroni, Anna; Siegelbaum, Steven

    2013-12-01

    Hyperpolarization-activated cyclic nucleotide-regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

  16. The Role of Type 4 Phosphodiesterases in Generating Microdomains of cAMP: Large Scale Stochastic Simulations

    PubMed Central

    Oliveira, Rodrigo F.; Terrin, Anna; Di Benedetto, Giulietta; Cannon, Robert C.; Koh, Wonryull; Kim, MyungSook; Zaccolo, Manuela; Blackwell, Kim T.

    2010-01-01

    Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons. PMID:20661441

  17. AmpG is required for BlaXc beta-lactamase expression in Xanthomonas campestris pv. campestris str. 17.

    PubMed

    Yang, Tsuey-Ching; Chen, Tzu-Fan; Tsai, Jeffrey J P; Hu, Rouh-Mei

    2013-03-01

    The chromosomal ampR(Xc) -bla(Xc) module is essential for the β-lactam resistance of Xanthomonas campestris pv. campestris. Bla(Xc) β-lactamase is expressed at a high basal level in the absence of an inducer and its expression can be further induced by β-lactam. In enterobacteria, ampG encodes an inner membrane facilitator involved in the recycling of murein degradation compounds. An isogenic ampG mutant (XcampG) of X. campestris pv. campestris str. 17 (Xc17) was constructed to investigate the link between murein recycling and bla(Xc) expression. Our data demonstrate that (1) XcampG is susceptible to β-lactam antibiotics; (2) AmpG(Xc) is essential for expression of bla(Xc) ; (3) AmpGs of Xc17, Stenotrophomonas maltophilia KJ (SmKJ) and Escherichia coli DH5α can complement the defect of XcampG; (4) overexpression of AmpG(X) (c) significantly increased bla(Xc) expression; and (5) AmpG(Xc) from Xc17 is able to restore β-lactamase induction of the ampN(Xc) -ampG(Xc) double mutant of SmKJ. In Xc17, ampG(Xc) can be expressed from the promoter residing in the intergenic region of ampN(Xc) -ampG(Xc) and the expression is independent of β-lactam induction. AmpN, which is required for β-lactamases induction in SmKJ, is not required for the β-lactam antibiotic resistance of Xc17.

  18. Forskolin's effect on transient K current in nudibranch neurons is not reproduced by cAMP.

    PubMed

    Coombs, J; Thompson, S

    1987-02-01

    Forskolin, a diterpene extracted from Coleus forskolii, stimulates the production of cAMP in a variety of cells and is potentially an important tool for studying the role of cAMP in the modulation of neuronal excitability. We studied the effects of forskolin on neurons of nudibranch molluscs and found that it caused characteristic, reversible changes in the amplitude and waveform of the transient K current, IA, and also activated an inward current similar to the cAMP-dependent inward current previously described in molluscan neurons. Forskolin altered the time course of IA activation and inactivation but did not affect the voltage dependence or the reversal potential of the current. IA normally inactivates exponentially, but in forskolin the time course of inactivation can be fit by the sum of 2 exponentials with an initial rate that is faster than the control and a final rate that is much slower. On depolarization in forskolin, IA begins to activate at the normal rate, but a slower component of activation is also seen. The changes in IA in the nudibranch cells were qualitatively different than the changes caused by forskolin in Aplysia bag cell neurons (Strong, 1984). Experiments were performed to determine whether these effects of forskolin require cAMP. Intracellular injection of cAMP, application of membrane-permeable analogs of cAMP, application of phosphodiesterase inhibitors, and intracellular injection of the active catalytic subunit of cAMP-dependent protein kinase did not affect the amplitude or waveform of IA. Also, the changes in IA that are caused by forskolin were not prevented or reversed by intracellular injection of an inhibitor of cAMP-dependent protein kinase. Cyclic AMP did, however, activate inward current at voltages near the resting potential. We conclude that the changes in IA and the activation of inward current represent separate affects of forskolin. The inward current appears to depend on an increase in intracellular cAMP, while the

  19. Atmospheric, Magnetospheric and Plasmas in Space (AMPS) spacelab payload definition study. Volume 3: Interface control documents. Part 3: AMPS payload to instruments ICD

    NASA Technical Reports Server (NTRS)

    1976-01-01

    General physical, functional, and operational interface control requirements for instruments on the first AMPS payload are presented. Interface specifications are included to satisfy ground handling, prelaunch, launch, stowage, operation, and landing activities. Applicable supporting documentation to implement the information is also given.

  20. Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP.

    PubMed

    Takahashi, Maho; Li, Yanping; Dillon, Tara J; Stork, Philip J S

    2017-01-27

    Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.

  1. The cAMP-producing agonist beraprost inhibits human vascular smooth muscle cell migration via exchange protein directly activated by cAMP

    PubMed Central

    McKean, Jenny S.; Murray, Fiona; Gibson, George; Shewan, Derryck A.; Tucker, Steven J.; Nixon, Graeme F.

    2015-01-01

    Aims During restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved. Methods and results In a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC. Conclusions These results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation. PMID:26092100

  2. Influence of cAMP on reporter bioassays for dioxin and dioxin-like compounds.

    PubMed

    Kasai, Ayumi; Yao, Jian; Yamauchi, Kozue; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Meng, Yiman; Maeda, Shuichiro; Kitamura, Masanori

    2006-02-15

    In reporter assays for detection of dioxins, the dioxin-responsive element (DRE) is generally used as a sensor sequence. In several systems, the CYP1A1 promoter containing DREs (DRE(cyp)) is inserted into a part of the long terminal repeat of mouse mammary tumor virus (LTR(MMTV)) to improve sensitivity of assays. We found that DRE(cyp)-LTR(MMTV) responds not only to dioxins and dioxin-like compounds but also to forskolin, a cAMP-elevating agent. This effect was dose-dependent and reproduced by other cAMP-elevating agents including 8-bromo-cAMP and 3-isobutyl-methylxanthine. The cAMP response element (CRE) and CRE-like sequences were absent in DRE(cyp)-LTR(MMTV) and not involved in this process. In contrast to the effect of dioxin, the activation of DRE(cyp)-LTR(MMTV) by cAMP was independent of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor for DRE. Furthermore, neither DRE(cyp), LTR(MMTV) nor the consensus sequence of DRE alone was activated in response to cAMP. These data elucidated for the first time that the combination of DRE(cyp) with LTR(MMTV) causes a peculiar response to cAMP and suggested that use of AhR antagonists is essential to exclude false-positive responses of DRE(cyp)-LTR(MMTV)-based bioassays for detection and quantification of dioxins and dioxin-like compounds.

  3. Depigmenting action of platycodin D depends on the cAMP/Rho-dependent signalling pathway.

    PubMed

    Jung, Eunsun; Hwang, Wangtaek; Kim, Seungbeom; Kim, Young-Soo; Kim, Yeong-Shik; Lee, Jongsung; Park, Deokhoon

    2011-12-01

    The overproduction and accumulation of melanin in the skin could lead to a pigmentary disorders, such as melasma, freckle, postinflammatory melanoderma and solar lentigo. Therefore, this study was conducted to investigate the effects of platycodin D (PD) on melanogenesis and its action mechanisms. In this study, we found that PD significantly inhibited melanin synthesis at low concentrations. These effects were further demonstrated by the PD-induced inhibition of cAMP production, phosphorylation of the cAMP-response element-binding protein and expression of microphthalmia-associated transcription factor and its downstream genes, tyrosinase, tyrosinase-related proteins-1 and Dct/tyrosinase-related proteins-2, suggesting that PD inhibits melanogenesis through the downregulation of cAMP signalling. Furthermore, PD induced significant morphological changes in melanocytes, namely, the retraction of dendrites. A small GTPase assays revealed that PD stimulated an increase in GTP-bound Rho content, one of downstream molecules of cAMP, but not in Rac or CDC42 content. Moreover, a Rho inhibitor (C3 exoenzyme) and a Rho kinase inhibitor (Y27632) attenuated the dendrite retraction induced by PD. Taken together, these findings indicate that PD inhibits melanogenesis by inhibiting the cAMP-protein kinase A pathway and also suppresses melanocyte dendricity through activation of the Rho signal that is mediated by PD-induced reduction in cAMP production. Therefore, these results suggest that PD exerts its inhibitory effects on melanogenesis and melanocyte dendricity via suppression of cAMP signalling and may be introduced as an inhibitor of hyperpigmentation caused by UV irradiation or pigmented skin disorders.

  4. Methionine adenosyltransferase:adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression.

    PubMed

    Kim, Jong-So; Coon, Steven L; Blackshaw, Seth; Cepko, Constance L; Møller, Morten; Mukda, Sujira; Zhao, Wan-Qian; Charlton, Clivel G; Klein, David C

    2005-01-07

    (S)-adenosylmethionine (SAM) is a critical element of melatonin synthesis as the methyl donor in the last step of the pathway, the O-methylation of N-acetyl 5-hydroxytryptamine by hydroxyindole-O-methyltransferase. The activity of the enzyme that synthesizes SAM, methionine adenosyltransferase (MAT), increases 2.5-fold at night in the pineal gland. In this study, we found that pineal MAT2A mRNA and the protein it encodes, MAT II, also increase at night, suggesting that the increase in MAT activity is caused by an increase in MAT II gene products. The night levels of MAT2A mRNA in the pineal gland were severalfold higher than in other neural and non-neural tissues examined, consistent with the requirement for SAM in melatonin synthesis. Related studies indicate that the nocturnal increase in MAT2A mRNA is caused by activation of a well described neural pathway that mediates photoneural-circadian regulation of the pineal gland. MAT2A mRNA and MAT II protein were increased in organ culture by treatment with norepinephrine (NE), the sympathetic neurotransmitter that stimulates the pineal gland at night. NE is known to markedly elevate pineal cAMP, and here it was found that cAMP agonists elevate MAT2A mRNA levels by increasing MAT2A mRNA synthesis and that drugs that block cAMP activation of cAMP dependent protein kinase block effects of NE. Therefore, the NE-cAMP dependent increase in pineal MAT activity seems to reflect an increase in MAT II protein, which occurs in response to cAMP-->protein kinase-dependent increased MAT2A expression. The existence of this MAT regulatory system underscores the importance that MAT plays in melatonin biogenesis. These studies also point to the possibility that SAM production in other tissues might be regulated through cAMP.

  5. G beta gamma signaling reduces intracellular cAMP to promote meiotic progression in mouse oocytes.

    PubMed

    Gill, Arvind; Hammes, Stephen R

    2007-02-01

    In nearly every vertebrate species, elevated intracellular cAMP maintains oocytes in prophase I of meiosis. Prior to ovulation, gonadotropins trigger various intra-ovarian processes, including the breakdown of gap junctions, the activation of EGF receptors, and the secretion of steroids. These events in turn decrease intracellular cAMP levels in select oocytes to allow meiotic progression, or maturation, to resume. Studies suggest that cAMP levels are kept elevated in resting oocytes by constitutive G protein signaling, and that the drop in intracellular cAMP that accompanies maturation may be due in part to attenuation of this inhibitory G protein-mediated signaling. Interestingly, one of these G protein regulators of meiotic arrest is the Galpha(s) protein, which stimulates adenylyl cyclase to raise intracellular cAMP in two important animal models of oocyte development: Xenopus leavis frogs and mice. In addition to G(alpha)(s), constitutive Gbetagamma activity similarly stimulates adenylyl cyclase to raise cAMP and prevent maturation in Xenopus oocytes; however, the role of Gbetagamma in regulating meiosis in mouse oocytes has not been examined. Here we show that Gbetagamma does not contribute to the maintenance of murine oocyte meiotic arrest. In fact, contrary to observations in frog oocytes, Gbetagamma signaling in mouse oocytes reduces cAMP and promotes oocyte maturation, suggesting that Gbetagamma might in fact play a positive role in promoting oocyte maturation. These observations emphasize that, while many general concepts and components of meiotic regulation are conserved from frogs to mice, specific differences exist that may lead to important insights regarding ovarian development in vertebrates.

  6. Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

    PubMed

    Wright, Peter T; Nikolaev, Viacheslav O; O'Hara, Thomas; Diakonov, Ivan; Bhargava, Anamika; Tokar, Sergiy; Schobesberger, Sophie; Shevchuk, Andrew I; Sikkel, Markus B; Wilkinson, Ross; Trayanova, Natalia A; Lyon, Alexander R; Harding, Sian E; Gorelik, Julia

    2014-02-01

    The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of β2-adrenergic receptor (β2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate β2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of β2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased β2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally compartmentalized β2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial β2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of β2AR and compartmentation of β2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.

  7. Influence of cAMP on reporter bioassays for dioxin and dioxin-like compounds

    SciTech Connect

    Kasai, Ayumi; Yao, Jian; Yamauchi, Kozue; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Meng, Yiman; Maeda, Shuichiro; Kitamura, Masanori . E-mail: masanori@yamanashi.ac.jp

    2006-02-15

    In reporter assays for detection of dioxins, the dioxin-responsive element (DRE) is generally used as a sensor sequence. In several systems, the CYP1A1 promoter containing DREs (DRE{sup cyp}) is inserted into a part of the long terminal repeat of mouse mammary tumor virus (LTR{sup MMTV}) to improve sensitivity of assays. We found that DRE{sup cyp}-LTR{sup MMTV} responds not only to dioxins and dioxin-like compounds but also to forskolin, a cAMP-elevating agent. This effect was dose-dependent and reproduced by other cAMP-elevating agents including 8-bromo-cAMP and 3-isobutyl-methylxanthine. The cAMP response element (CRE) and CRE-like sequences were absent in DRE{sup cyp}-LTR{sup MMTV} and not involved in this process. In contrast to the effect of dioxin, the activation of DRE{sup cyp}-LTR{sup MMTV} by cAMP was independent of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor for DRE. Furthermore, neither DRE{sup cyp}, LTR{sup MMTV} nor the consensus sequence of DRE alone was activated in response to cAMP. These data elucidated for the first time that the combination of DRE{sup cyp} with LTR{sup MMTV} causes a peculiar response to cAMP and suggested that use of AhR antagonists is essential to exclude false-positive responses of DRE{sup cyp}-LTR{sup MMTV}-based bioassays for detection and quantification of dioxins and dioxin-like compounds.

  8. Replenishing the cyclic-di-AMP pool: regulation of diadenylate cyclase activity in bacteria.

    PubMed

    Pham, Thi Huong; Liang, Zhao-Xun; Marcellin, Esteban; Turner, Mark S

    2016-11-01

    Bacteria can sense environmental cues and alter their physiology accordingly through the use of signal transduction pathways involving second messenger nucleotides. One broadly conserved second messenger is cyclic-di-AMP (c-di-AMP) which regulates a range of processes including cell wall homeostasis, potassium uptake, DNA repair, fatty acid synthesis, biofilm formation and central metabolism in bacteria. The intracellular pool of c-di-AMP is maintained by the activities of diadenylate cyclase (DAC) and phosphodiesterase (PDE) enzymes, as well as possibly via c-di-AMP export. Whilst extracellular stimuli regulating c-di-AMP levels in bacteria are poorly understood, recent work has identified effector proteins which directly interact and alter the activity of DACs. These include the membrane bound CdaR and the phosphoglucosamine mutase GlmM which both bind directly to the membrane bound CdaA DAC and the recombination protein RadA which binds directly to the DNA binding DisA DAC. The genes encoding these multiprotein complexes are co-localised in many bacteria providing further support for their functional connection. The roles of GlmM in peptidoglycan synthesis and RadA in Holliday junction intermediate processing suggest that c-di-AMP synthesis by DACs will be responsive to these cellular activities. In addition to these modulatory interactions, permanent dysregulation of DAC activity due to suppressor mutations can occur during selection to overcome growth defects, rapid cell lysis and osmosensitivity. DACs have also been investigated as targets for the development of new antibiotics and several small compound inhibitors have recently been identified. This review aims to provide an overview of how c-di-AMP synthesis by DACs can be regulated.

  9. The β-lactamase inhibitor avibactam (NXL104) does not induce ampC β-lactamase in Enterobacter cloacae

    PubMed Central

    Miossec, Christine; Claudon, Monique; Levasseur, Premavathy; Black, Michael T

    2013-01-01

    Induction of ampC β-lactamase expression can often compromise antibiotic treatment and is triggered by several β-lactams (such as cefoxitin and imipenem) and by the β-lactamase inhibitor clavulanic acid. The novel β-lactamase inhibitor avibactam (NXL104) is a potent inhibitor of both class A and class C enzymes. The potential of avibactam for induction of ampC expression in Enterobacter cloacae was investigated by ampC messenger ribonucleic acid quantitation. Cefoxitin and clavulanic acid were confirmed as ampC inducers, whereas avibactam was found to exert no effect on ampC expression. Thus, avibactam is unlikely to diminish the activity of any partner β-lactam antibiotic against AmpC-producing organisms. PMID:24348054

  10. Glucose and GLP-1 Stimulate cAMP Production via Distinct Adenylyl Cyclases in INS-1E Insulinoma Cells

    PubMed Central

    Ramos, Lavoisier S.; Zippin, Jonathan Hale; Kamenetsky, Margarita; Buck, Jochen; Levin, Lonny R.

    2008-01-01

    In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades. PMID:18695009

  11. Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.

    PubMed

    Odaka, Haruki; Arai, Satoshi; Inoue, Takafumi; Kitaguchi, Tetsuya

    2014-01-01

    Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.

  12. Outward currents in Drosophila larval neurons: dunce lacks a maintained outward current component downregulated by cAMP.

    PubMed

    Delgado, R; Davis, R; Bono, M R; Latorre, R; Labarca, P

    1998-02-15

    Outward current modulation by cAMP was investigated in wild type (wt) and dunce (dnc) Drosophila larval neurons. dnc is deficient in a cAMP phosphodiesterase and has altered memory. Outward current modulation by cAMP was investigated by acute or chronic exposure to cAMP analogs. The analysis included a scrutiny of outward current modulation by cAMP in neurons from the mushroom bodies (mrb). In Drosophila, the mrb are the centers of olfactory acquisition and retention. Based on outward current patterns, neurons were classified into four types. Downmodulation of outward currents induced by acute application of cAMP analogs was reversible and found only in type I and type IV neurons. In the general wt neuron population, approximately half of neurons exhibited cAMP-modulated, 4-aminopyridine (4-AP)-sensitive currents. On the other hand, a significantly larger fraction of mrb neurons in wt (70%) was endowed with cAMP-modulated, 4-AP-sensitive currents. Only 30% of the dnc neurons displayed outward currents modulated by cAMP. The deficit of cAMP-modulated outward currents was most severe in neurons derived from the mrb of dnc individuals. Only 4% of the mrb neurons of dnc were cAMP-modulated. The dnc defect can be induced by chronic exposure of wt neurons to cAMP analogs. These results document for the first time a well defined electrophysiological neuron phenotype in correlation with the dnc defect. Moreover, this study demonstrates that in dnc mutants such a deficiency affects most severely neurons in brain centers of acquisition and retention.

  13. The regulation of chemotaxis and chemokinesis in Dictyostelium amoebae by temporal signals and spatial gradients of cyclic AMP.

    PubMed

    Vicker, M G

    1994-02-01

    The tactic and kinetic locomotion of Dictyostelium discoideum amoebae were examined in cyclic AMP (cAMP) spatial gradient and temporal signal fields. The distributions of migrating cells were examined within 150 microns-thick micropore filters after incubation with different cAMP concentrations, [cAMP], applied in three ways across the fields: as positively or negatively developing gradients, generated either by increasing or decreasing the [cAMP] on one side of the filter, respectively, or as static, linear gradients after negative development. Chemotaxis was only induced by oriented, temporally increasing [cAMP]. Pulses propagated by molecular diffusion or mechanical flow were equally effective. Negatively developing cAMP gradients had no initial effect on cell accumulation. However, if the subsequent static spatial gradient was maintained by an infusion system, some gradients also induced cell accumulation, whose degree and direction depended on the gradient [cAMP]. The basis of this new effect was examined by tracking individual cells by computer-assisted videomicroscopy during locomotion in different [cAMP]. Cells produced a triphasic [cAMP]-dependent response, with optimal cell motility induced by 10-30 nM. The results demonstrate that cell accumulation either up-field or down-field in spatial gradients is governed by the field locations of the attractant concentrations that induce the relative locomotory maxima and minima in the gradient field. Cells perceive the ambient [cAMP], but cannot read the spatial gradient orientation in static or yet steeper regions of developing gradients. Accumulation in static spatial gradients is a function of klino- and orthokinesis, but chemotaxis requires an oriented cAMP pulse or impulse.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

    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.

  15. cAMP Promotes Cell Migration Through Cell Junctional Complex Dynamics and Actin Cytoskeleton Remodeling: Implications in Skin Wound Healing.

    PubMed

    Kim, Mi Ok; Ryu, Jung Min; Suh, Han Na; Park, Soo Hyun; Oh, Yeon-Mok; Lee, Sang Hun; Han, Ho Jae

    2015-11-01

    Stem cells have attracted great interest for their therapeutic capacity in tissue regeneration. Cyclic adenosine 3',5'-monophosphate (cAMP), existing in high concentration at wound sites, mediated various signaling pathways such as cytoskeleton dynamics, cell adhesion, and cell migration in stem cells, which suggest the critical roles of cAMP in the wound healing process through functional regulation of stem cells. However, the mechanisms behind the effect of cAMP on mouse embryonic stem cell (mESC) motility and its roles on skin wound healing remain to be fully elucidated. In the present study, 8-Bromo cAMP-treated mESCs showed significant wound closure and improved neovascularization. Moreover, 8-Bromo cAMP stimulated mESC migration into the wound bed. 8-Bromo cAMP also increased ESC motility in in vitro migration assay. 8-Bromo cAMP induced myosin light chain phosphorylation through Rac1 and Cdc42 signaling, which were involved in 8-Bromo cAMP-induced decrease in expression of junction proteins (connexin 43, E-cadherin, and occludin) at the plasma membrane. Subsequently, 8-Bromo cAMP induced the disruption of cell junctions (including gap junctions, adherens junctions, and tight junctions), which reduced the function of the gap junctions and cell adhesion. In addition, 8-Bromo cAMP-induced Rac1 and Cdc42 activation increased Arp3, TOCA, PAK, and N-WASP expression, but decreased cofilin phosphorylation level, which elicited actin cytoskeleton remodeling. In contrast to the control, 8-Bromo cAMP evoked a substantial migration of cells into the denuded area, which was blocked by the small interfering RNAs of the signaling pathway-related molecules or by inhibitors. In conclusion, cAMP enhanced the migration of mESCs through effective coordination of junctional disruption and actin cytoskeleton remodeling, which increased the wound healing capacity of ESCs.

  16. A jack of all trades: the multiple roles of the unique essential second messenger cyclic di-AMP.

    PubMed

    Commichau, Fabian M; Dickmanns, Achim; Gundlach, Jan; Ficner, Ralf; Stülke, Jörg

    2015-07-01

    Second messengers are key components of many signal transduction pathways. In addition to cyclic AMP, ppGpp and cyclic di-GMP, many bacteria use also cyclic di-AMP as a second messenger. This molecule is synthesized by distinct classes of diadenylate cyclases and degraded by phosphodiesterases. The control of the intracellular c-di-AMP pool is very important since both a lack of this molecule and its accumulation can inhibit growth of the bacteria. In many firmicutes, c-di-AMP is essential, making it the only known essential second messenger. Cyclic di-AMP is implicated in a variety of functions in the cell, including cell wall metabolism, potassium homeostasis, DNA repair and the control of gene expression. To understand the molecular mechanisms behind these functions, targets of c-di-AMP have been identified and characterized. Interestingly, c-di-AMP can bind both proteins and RNA molecules. Several proteins that interact with c-di-AMP are required to control the intracellular potassium concentration. In Bacillus subtilis, c-di-AMP also binds a riboswitch that controls the expression of a potassium transporter. Thus, c-di-AMP is the only known second messenger that controls a biological process by interacting with both a protein and the riboswitch that regulates its expression. Moreover, in Listeria monocytogenes c-di-AMP controls the activity of pyruvate carboxylase, an enzyme that is required to replenish the citric acid cycle. Here, we review the components of the c-di-AMP signaling system.

  17. 5-HT induces cAMP production in crypt colonocytes at a 5-HT4 receptor.

    PubMed

    Albuquerque, F C; Smith, E H; Kellum, J M

    1998-07-01

    Previous studies demonstrate that both 5-hydroxytryptamine (5-HT) and cyclic AMP (cAMP) induce chloride efflux from crypt colonocytes in the rat distal colon; antagonist studies suggest that the 5-HT response is mediated primarily by the 5-HT4 receptor. Since this receptor is known to be positively coupled to adenylate cyclase, we postulated that 5-HT should induce generation of cAMP, which should be inhibited by 5-HT4 antagonists. Method. Mucosal cells from rat distal colon were taken by a sequential calcium chelation technique for enrichment of crypt cells. Cytokeratin stains demonstrated that >99% of cells were colonocytes. [3H]Thymidine uptake studies demonstrate a fivefold increased incorporation in this cell preparation compared to earlier fractions. 3-Isobutyl-l-methylxanthine (IBMX, 100 microM) was added to all cell suspensions in order to prevent cAMP metabolism. Cell suspensions were incubated for 2 min at 37 degreesC with different concentrations of 5-HT (n = 7). cAMP was measured by enzyme immunoassay. In another series of experiments, 5-HT (0.3 microM) stimulation of cAMP was similarly measured in the presence and absence of 5-HT receptor antagonists: 10 microM 5-HTP-DP (5-HT1P; n = 4), 0.1 microM ketanserin (5-HT2A; n = 4), 0.3 microM ondansetron (5-HT3; n = 4), 3 microM tropisetron (5-HT3 and 5-HT4; n = 4), and 10 nM GR-113808 (5-HT4; n = 5). Results. 5-HT produced a dose-dependent increase in cAMP. The increase was significant at concentrations >/=0.3 microM when compared to cells incubated with IBMX alone. In the second series of experiment, 5-HT-induced generation of cAMP at a dose of 0.3 microM was significantly inhibited in the presence of GR-113808 and tropisetron. Conclusion. 5-HT acts at a 5-HT4 receptor to induce production of cAMP in rat distal crypt colonocytes.

  18. Activation of cyclic amp/protein kinase: a signaling pathway enhances osteoblast cell adhesion on biomaterials for regenerative engineering.

    PubMed

    Lo, Kevin W-H; Ashe, Keshia M; Kan, Ho Man; Lee, Duron A; Laurencin, Cato T

    2011-04-01

    Osteoblast cell adhesion on biomaterials is an important goal for implants to be useful in bone regeneration technologies. The adhesion of osteoblastic cells to biomaterials has been investigated in the field of bone regenerative engineering. Previous work from our group demonstrated that osteoblastic cells adhering to biodegradable biomaterials require the expression of integrins on the cell surface. However, the underlying molecular signaling mechanism is still not fully clear. We report here that cyclic adenosine monophosphate (cAMP), a small signaling molecule, regulates osteoblast cell adhesion to biomaterial surfaces. We used an in vitro cell adhesion assay to demonstrate that at 0.1 mM, 8-Br-cAMP, a cell-permeable cAMP analog, significantly enhances osteoblast-like cells' (MC3T3-E1) adherence to biomaterials. Moreover, we demonstrate that a commonly used cAMP-elevating agent, forskolin, promotes cell adhesion similar to that of the cell permeable cAMP analog. By using different target-specific cAMP analogs: 8-CPT-2Me-cAMP which specifically activates exchange protein activated by cAMP (Epac), and 6-Bnz-cAMP which specifically activates protein kinase A (PKA), we observed that the PKA signaling pathway plays a dominant role in this process. Thus, this report suggests a new method to enhance osteoblast cell adhesion on biodegradable biomaterials for bone regenerative engineering applications.

  19. Cyclic adenosine monophosphate (cAMP)-induced histone hyperacetylation contributes to its antiproliferative and differentiation-inducing activities.

    PubMed

    Yoo, Seungwan; Lee, Yong Gyu; Kim, Ji Hye; Byeon, Se Eun; Rho, Ho Sik; Cho, Jae Youl; Hong, Sungyoul

    2012-01-01

    Histone acetylation is linked to the control of chromatin remodeling, which is involved in cell growth, proliferation, and differentiation. It is not fully understood whether cyclic adenosine monophosphate (cAMP), a representative differentiation-inducing molecule, is able to modulate histone acetylation as part of its anticancer activity. In the present study, we aimed to address this issue using cell-permeable cAMP, i.e. dibutyryl cAMP (dbcAMP) and C6 glioma cells. As reported previously, under the conditions of our studies, treatment with dbcAMP clearly arrested C6 cell proliferation and altered their morphology. Its antiproliferative and differentiation-inducing activity in C6 glioma cells involved upregulation of p219WAF/CIP), p27(kip1), glial fibrillary acidic protein (GFAP), and Cx43, as well as downregulation of vimentin. Furthermore, dbcAMP modulated the phosphorylation of ERK and Akt in a time-dependent manner and altered the colocalization pattern of phospho-Src and the actin cytoskeleton. Interestingly, dbcAMP upregulated the enzyme activity of histone acetyltransferase (HAT) and, in parallel, enhanced cellular acetyllysine levels. Finally, the hyperacetylation-inducing compound, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, displayed similar anticancer activity to dbcAMP. Therefore, our data suggest that antiproliferative and differentiation-inducing activities of dbcAMP may be generated by its enhanced hyperacetylation function.

  20. Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    PubMed Central

    Almeida, Luis E. F.; Murray, Peter D.; Zielke, H. Ronald; Roby, Clinton D.; Kingsbury, Tami J.; Krueger, Bruce K.

    2009-01-01

    Cyclic AMP can stimulate the transcription of many activity-dependent genes via activation of the transcription factor, CREB. However, in mouse cortical neuron cultures, prior to synaptogenesis, neither cAMP nor dopamine, which acts via cAMP, stimulated CREB-dependent gene transcription when NR2B-containing NMDA receptors (NMDARs) were blocked. Stimulation of transcription by cAMP was potentiated by inhibitors of excitatory amino acid uptake, suggesting a role for extracellular glutamate or aspartate in cAMP-induced transcription. Aspartate was identified as the extracellular messenger: enzymatic scavenging of L-aspartate, but not glutamate, blocked stimulation of CREB-dependent gene transcription by cAMP; moreover, cAMP induced aspartate but not glutamate release. Taken together, these results suggest that cAMP acts via an autocrine or paracrine pathway to release aspartate, which activates NR2B-containing NMDARs, leading to Ca2+ entry and activation of transcription. This cAMP/aspartate/NMDAR signaling pathway may mediate the effects of transmitters such as dopamine on axon growth and synaptogenesis in developing neurons or on synaptic plasticity in mature neural networks. PMID:19812345

  1. [Isolation and study of the properties of the regulator subunit of cAMP-dependent protein kinase].

    PubMed

    Iurkiv, V A; Severin, E S; Petukhov, S P; Bulargina, T V

    1982-12-01

    The regulatory subunit of type II cAMP-dependent proteinkinase was isolated from cytosol of the rabbit small intestinal mucosa by affinity chromatography. The preparation contained 3 proteolytic enzymes and occurred in two forms differing as regards cAMP affinity. The cAMP-binding capacity of the preparation was equal to 17 nmol cAMP/mg protein. To study the topography of the cAMP-binding center, use was made of cAMP analogs. It was demonstrated that introduction of the substituents into the 8th position of the purine ring and substitution with respect to the N6-exoaminogroup affected insignificantly the analog affinity for the cAMP-binding center. At the same time the substituents introduced into the first position of the adenine base, into the area of the 2'-hydroxyl group of ribose and into the cyclophosphate part of the cAMP molecule considerably decreased the analog affinity for the regulatory center of type II cAMP-dependent proteinkinase.

  2. Innate hemocyte responses of Malacosoma disstria larvae (C. Insecta) to antigens are modulated by intracellular cyclic AMP.

    PubMed

    Gulii, Vladislav; Dunphy, Gary B; Mandato, Craig A

    2009-08-01

    Invertebrate intracellular hemocyte signaling pathways affecting cellular-antigen responses, although defined for molluscs and some arthropods including dipteran insects, is less known for lepidopterans. Hemocytic-antigen responses of the arboreal pest lepidopteran Malacosoma disstria are linked to cAMP-dependent protein kinase A implicating cAMP in cellular hemocyte immune responses. The purpose in the present study was to determine intracellular cAMP effects on larval M. disstria hemocytes adhering to slides and bacteria. Altering adenylate cyclase and phosphodiesterase activities as well as cAMP levels in vitro and in vivo changed hemocyte responses to antigens. Quiescent hemocytes had high cAMP levels due to adenylate cyclase activity and possibly low phosphodiesterase (type 4) activity. Antigen contact diminished hemocytic cAMP levels. Inhibiting adenylate cyclase increased hemocyte-antigen and hemocyte-hemocyte adhesion, the latter producing nodules in vivo without bacterial antigens. Inhibiting phosphodiesterase type 4 produced the reverse effects. Pharmacologically increasing intracellular cAMP in attached hemocytes caused many of the cells to detach. Diminished intracellular cAMP changed hemograms in vivo in bacteria-free larvae comparable to changes induced by the bacterium, Bacillus subtilis, by producing nodules. Lowering cAMP enhanced also the removal of Xenorhabdus nematophila and B. subtilisin vivo.

  3. Intraocular elevation of cyclic AMP potentiates ciliary neurotrophic factor-induced regeneration of adult rat retinal ganglion cell axons.

    PubMed

    Cui, Qi; Yip, Henry K; Zhao, Robert C H; So, Kwok-Fai; Harvey, Alan R

    2003-01-01

    In vitro, cyclic AMP (cAMP) elevation alters neuronal responsiveness to diffusible growth factors and myelin-associated inhibitory molecules. Here we used an established in vivo model of adult central nervous system injury to investigate the effects of elevated cAMP on neuronal survival and axonal regeneration. We studied the effects of intraocular injections of neurotrophic factors and/or a cAMP analogue (CPT-cAMP) on the regeneration of axotomized rat retinal ganglion cell (RGC) axons into peripheral nerve autografts. Elevation of cAMP alone did not significantly increase RGC survival or the number of regenerating RGCs. Ciliary neurotrophic factor increased RGC viability and axonal regrowth, the latter effect substantially enhanced by coapplication with CPT-cAMP. Under these conditions over 60% of surviving RGCs regenerated their axons. Neurotrophin-4/5 injections also increased RGC viability, but there was reduced long-distance axonal regrowth into grafts, an effect partially ameliorated by cAMP elevation. Thus, cAMP can act cooperatively with appropriate neurotrophic factors to promote axonal regeneration in the injured adult mammalian central nervous system.

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

    SciTech Connect

    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.

  5. The central role of cAMP in regulating Plasmodium falciparum merozoite invasion of human erythrocytes.

    PubMed

    Dawn, Amrita; Singh, Shailja; More, Kunal R; Siddiqui, Faiza Amber; Pachikara, Niseema; Ramdani, Ghania; Langsley, Gordon; Chitnis, Chetan E

    2014-12-01

    All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.

  6. Retromer terminates the generation of cAMP by internalized PTH-receptors

    PubMed Central

    Feinstein, Timothy N.; Wehbi, Vanessa L.; Ardura, Juan; Wheeler, David S.; Ferrandon, Sebastien; Gardella, Thomas J.; Vilardaga, Jean-Pierre

    2011-01-01

    Generation of cAMP by G protein–coupled receptors (GPCRs) and its termination is currently thought to occur exclusively at the plasma membrane of cells. Under existing models of receptor regulation, this signal is primarily restricted by desensitizationof the receptors through their binding to β-arrestins. However, this paradigm is not consistent with recent observations that the parathyroid hormone receptor type 1 (PTHR) continues to stimulate cAMP production even after receptor internalization, as β-arrestins are known to rapidly bind and internalize activated PTHR. Here we show that β-arrestin1 binding prolongs rather than terminates cAMP generation by PTHR, and that cAMP generation correlates with the persistence of arrestin-receptor complexes on endosomes. We found that PTHR signaling is instead turned-off by the retromer complex, which regulates traffic of internalized receptor from endosomes to the Golgi apparatus. Thus, binding by the retromer complex regulates sustained cAMP generation triggered by an internalized GPCR. PMID:21445058

  7. Regulation of rhythmic melatonin production in pineal cells of chick embryo by cyclic AMP.

    PubMed

    Macková, M; Lamosová, D; Zeman, M

    1998-05-01

    The pineal cells of chick embryos incubated in vitro exhibited a daily rhythm of melatonin synthesis under a 12:12 light:dark (LD) cycle at the embryonic days 16 and 19. In order to elucidate whether cyclic adenosine monophosphate (cAMP)--a component of the melatonin generating system--is already at work in the embryonic period, we measured the effects of forskolin and isobuthylmethylxantine (IBMX) on melatonin production, cAMP efflux and accumulation. Forskolin (after 10, 20, 30, 45, 60 and 90 min of administration) and IBMX (6 h), when applied during the light phase of LD cycle, stimulated melatonin production and cAMP efflux and accumulation during the embryonic period (at days 16 and 19 fo development). Our results suggest that the biochemical pathway involving cAMP, which controls melatonin production in the postnatal period, is developed before hatching and already on embryonic day 19 works in a way similar to that in post-hatched chicks. Differences in response to cAMP stimulation between 16- and 19-day-old pinealocytes seem to be mostly quantitative.

  8. cAMP regulates axon outgrowth and guidance during optic nerve regeneration in goldfish.

    PubMed

    Rodger, J; Goto, H; Cui, Q; Chen, P B; Harvey, A R

    2005-11-01

    Increased cAMP improves neuronal survival and axon regeneration in mammals. Here, we assess cAMP levels and identify activated pathways in a spontaneously regenerating central nervous system. Following optic nerve crush in goldfish, almost all retinal ganglion cells (RGC) survive and regenerate retinotectal topography. Goldfish received injections of a cAMP analogue (CPT-cAMP), a protein kinase A (PKA) inhibitor (KT5720), both compounds combined, or PBS (control). RGC survival in experimental groups was unaffected at any stage. The rate of axon regeneration was accelerated by the activator and decelerated both by the inhibitor and by combined injections, suggesting a PKA-dependent pathway. In addition, errors in regenerate retinotectal topography were observed when agents were applied in vivo and RGC response to the guidance cue ephrin-A5 in vitro was altered by the inhibitor. Our results highlight that therapeutic manipulation of cAMP levels to enhance axonal regeneration in mammals must ensure that topography, and consequently function, is not disrupted.

  9. Cyclic AMP, Protein Kinase A, and Phosphodiesterases: Proceedings of an International Workshop

    PubMed Central

    Stratakis, C. A.

    2014-01-01

    Cyclic nucleotides cAMP and cGMP are part of almost all major cellular signaling pathways. Phosphodiesterases (PDEs) are enzymes that regulate the intracellular levels of cAMP and cGMP. Protein kinase A or cAMP-dependent protein kinase mediates most cAMP effects in the cell. Over the last 25 years, various components of this group of molecules have been involved in human diseases, both genetic and acquired. Lately, the PDEs attract more attention. The pharmacological exploitation of the PDE’s ability to regulate cGMP and cAMP, and through them, a variety of signaling pathways, has led to a number of new drugs for diverse applications from the treatment of erectile dysfunction to heart failure, asthma, and chronic obstructive pulmonary disease. We present the abstracts (available online) and selected articles from the proceedings of a meeting that took place at the National Institutes of Health (NIH), Bethesda, MD, June 8–10, 2011. PMID:22951901

  10. Modulation of cAMP metabolism in Mycobacterium tuberculosis and its effect on host infection.

    PubMed

    Barba, Jeannette; Alvarez, Angel H; Flores-Valdez, Mario Alberto

    2010-05-01

    Mycobacterium tuberculosis remains the single most relevant bacterial infectious agent as Tuberculosis is estimated to affect one-third of the world population. Like other microorganisms, M. tuberculosis needs to sense and adapt to changes in the several niches where it is found, ranging from the environment to a number of host-adapted programs, including infection of cell types such as macrophages, dendritic cells, epithelial cells and adipocytes. A strategy commonly used by cells to respond to such changes consists of producing small molecules known as second messengers. 3',5'-cyclic adenosine monophosphate (cAMP) is one of the best-studied second messengers in many organisms, and in recent years its participation during the M. tuberculosis infection cycle has just begun to be thoroughly considered. In this work, we aimed to provide a perspective of how cAMP metabolism proceeds in M. tuberculosis, which genes are activated in response to cAMP signaling in this organism, and discuss the evidence for bacterially produced cAMP use during infection. Furthermore, key issues needing to be addressed for better understanding cAMP physiology in slow-growing pathogenic mycobacteria are presented.

  11. cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

    PubMed Central

    Bodor, J; Spetz, A L; Strominger, J L; Habener, J F

    1996-01-01

    Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T-lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I. Images Fig. 3 Fig. 4 Fig. 5 PMID:8622971

  12. Epac2 Mediates cAMP-Dependent Potentiation of Neurotransmission in the Hippocampus.

    PubMed

    Fernandes, Herman B; Riordan, Sean; Nomura, Toshihiro; Remmers, Christine L; Kraniotis, Stephen; Marshall, John J; Kukreja, Lokesh; Vassar, Robert; Contractor, Anis

    2015-04-22

    Presynaptic terminal cAMP elevation plays a central role in plasticity at the mossy fiber-CA3 synapse of the hippocampus. Prior studies have identified protein kinase A as a downstream effector of cAMP that contributes to mossy fiber LTP (MF-LTP), but the potential contribution of Epac2, another cAMP effector expressed in the MF synapse, has not been considered. We investigated the role of Epac2 in MF-CA3 neurotransmission using Epac2(-/-) mice. The deletion of Epac2 did not cause gross alterations in hippocampal neuroanatomy or basal synaptic transmission. Synaptic facilitation during short trains was not affected by loss of Epac2 activity; however, both long-term plasticity and forskolin-mediated potentiation of MFs were impaired, demonstrating that Epac2 contributes to cAMP-dependent potentiation of transmitter release. Examination of synaptic transmission during long sustained trains of activity suggested that the readily releasable pool of vesicles is reduced in Epac2(-/-) mice. These data suggest that cAMP elevation uses an Epac2-dependent pathway to promote transmitter release, and that Epac2 is required to maintain the readily releasable pool at MF synapses in the hippocampus.

  13. cAMP-activated chloride currents in amphibian retinal pigment epithelial cells.

    PubMed Central

    Hughes, B A; Segawa, Y

    1993-01-01

    1. The effect of cAMP on whole-cell currents in isolated retinal pigment epithelial (RPE) cells of the bullfrog and marine toad was investigated by means of the perforated patch clamp technique. 2. Superfusing cells with either cAMP or forskolin led to the development of a time-independent current that had a linear current-voltage (I-V) relationship. The reversal potential of (Vrev) of the cAMP-activated current was unaffected by the removal of either Na+ or HCO3- from the external and internal solutions or by the addition of extracellular barium, but it was near the Cl- equilibrium potential (ECl) over a wide range of extracellular Cl- concentrations, suggesting the presence of a Cl(-)-selective channel. 3. The anion permeability sequence of the cAMP-activated conductance calculated from biionic reversal potentials was NO3- = I- > Br- > Cl- >> HCO3- > methanesulphonate. 4. The conductance was blocked by a variety of Cl- transport inhibitors, including 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), 4,4'-dinitro-2,2'- stilbene disulphonic acid (DNDS), frusemide, N-phenylanthranilic acid (DPC) and niflumic acid. 5. The present study demonstrates that cAMP activates a Cl(-)-selective channel that most probably resides in the basolateral membrane. PMID:8410715

  14. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

    NASA Technical Reports Server (NTRS)

    Kandasamy, S. B.; Williaes, B. A.

    1983-01-01

    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

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

    PubMed

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

    2008-08-07

    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.

  16. Cyclic AMP restores a normal phenotype to sis oncogene transformed cells and inhibits inositol phospholipid turnover

    SciTech Connect

    Murphy, S.K.; Lazarus, A.; Pendergas, M.; Lockwood, A.H.

    1987-05-01

    The sis oncogene encodes the A chain of platelet-derived growth factor (PDGF). NIH3T3 fibroblasts transfected with the cloned sis oncogene display a malignant phenotype and have enhanced turnover of the regulatory phospholipid phosphatidylinositol 4,5 biphosphate (PIP2). They have found that elevation of intracellular cyclic AMP can restore many aspects of normal growth and morphology to sis-transformed cells. Cells rapidly become less refractile, flatten on the substratum, develop actomyosin bundles, and acquire a more tranquil membrane. Growth rate and saturation density are reduced. Cultures become contact-inhibited and, at confluence, assume a normal fibrobastic morphology. The ability to grow in low serum or suspension is lost. Following addition of 8-Br-cAMP, cellular levels of PIP and PIP2 increase to those in untransformed cells. Concurrently, the steady-state levels of inositol phosphates are reduced to normal values. They have found a similar effect of cAMP on inositol phospholipid metabolism in cells transformed by the human H-ras oncogene. These results suggest that cAMP, acting through the cAMP-dependent protein kinase, antagonizes ras and sis oncogene expression by inhibiting polyphosphoinositide turnover. Such action might occur by phosphorylation of the PDGF (sis) receptor or of a ras-stimulated phospholipase C.

  17. Identification of ATP diphosphohydrolase activity in human term placenta using a novel assay for AMP.

    PubMed

    Papamarcaki, T; Tsolas, O

    1990-09-03

    Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN3 were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase. A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05-0.8 microM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates. The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.

  18. Renal Epithelial Cyst Formation and Enlargement in vitro: Dependence on cAMP

    NASA Astrophysics Data System (ADS)

    Mangoo-Karim, Roberto; Uchic, Marie; Lechene, Claude; Grantham, Jared J.

    1989-08-01

    Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.

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

    SciTech Connect

    Lewis, E.J.; Harrington, C.A.; Chikaraishi, D.M.

    1987-06-01

    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 GH/sub 4/ 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.

  20. Evidence for cAMP as a mediator of gonadotropin secretion from female pituitaries

    SciTech Connect

    Bourne, G.A.; Baldwin, D.M.

    1987-09-01

    Sodium flufenamate, which inhibited gonadotropin-releasing hormone (GnRH)-stimulated increases in adenosine 3',5'-cyclic monophosphate (cAMP), was used to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion. Quartered pituitaries from diestrous II female rats were perifused at 37/sup 0/C, and sequential effluent fractions were collected every 10 min. Administration of GnRH resulted in a characteristic biphasic response for both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whereas 5 ..mu..M cycloheximide inhibited the secondary augmented responses (phase II) of both hormones. Infusions of 0.1 mM flufenamate inhibited GnRH-stimulated gonadotropin secretion in a manner similar to that of cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate resulted in the restoration of LH and FSH secretion. The dibutyryl cAMP-restored response appeared to be protein synthesis dependent and specific for cAMP. These results suggest that although the cyclic nucleotide is not involved in the acute release of LH and FSH, it does appear to play a pivotal but indirect role in phase II release of the hormones, by effects involving the stimulation of de novo protein synthesis.

  1. The role of ventral striatal cAMP signaling in stress-induced behaviors

    PubMed Central

    Plattner, Florian; Hayashi, Kanehiro; Hernandez, Adan; Benavides, David R.; Tassin, Tara C.; Tan, Chunfeng; Day, Jonathan; Fina, Maggy W.; Yuen, Eunice Y.; Yan, Zhen; Goldberg, Matthew S.; Nairn, Angus C.; Greengard, Paul; Nestler, Eric J.; Taussig, Ronald; Nishi, Akinori; Houslay, Miles D.; Bibb, James A.

    2015-01-01

    The cAMP/PKA signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitates cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targets the regulation of PDE4 by Cdk5, all produced analogical effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression. PMID:26192746

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

    PubMed Central

    Thompson, Eloise; Breil, Florence; Lorthiois, Audrey; Dupuy, Florian; Cummings, Ross; Duffier, Yoann; Corbett, Yolanda; Mercereau-Puijalon, Odile; Vernick, Kenneth; Taramelli, Donatella; Baker, David A.; Langsley, Gordon; Lavazec, Catherine

    2015-01-01

    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. PMID:25951195

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

    NASA Astrophysics Data System (ADS)

    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.

  4. Pseudomonas aeruginosa AmpR: an acute–chronic switch regulator

    PubMed Central

    Balasubramanian, Deepak; Kumari, Hansi; Mathee, Kalai

    2015-01-01

    Pseudomonas aeruginosa is one of the most intractable human pathogens that pose serious clinical challenge due to extensive prevalence of multidrug-resistant clinical isolates. Armed with abundant virulence and antibiotic resistance mechanisms, it is a major etiologic agent in a number of acute and chronic infections. A complex and intricate network of regulators dictates the expression of pathogenicity factors in P. aeruginosa. Some proteins within the network play key roles and control multiple pathways. This review discusses the role of one such protein, AmpR, which was initially recognized for its role in antibiotic resistance by regulating AmpC β-lactamase. Recent genomic, proteomic and phenotypic analyses demonstrate that AmpR regulates expression of hundreds of genes that are involved in diverse pathways such as β-lactam and non-β-lactam resistance, quorum sensing and associated virulence phenotypes, protein phosphorylation, and physiological processes. Finally, ampR mutations in clinical isolates are reviewed to shed light on important residues required for its function in antibiotic resistance. The prevalence and evolutionary implications of AmpR in pathogenic and nonpathogenic proteobacteria are also discussed. A comprehensive understanding of proteins at nodal positions in the P. aeruginosa regulatory network is crucial in understanding, and ultimately targeting, the pathogenic stratagems of this organism. PMID:25066236

  5. Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

    PubMed

    Solursh, M; Reiter, R; Ahrens, P B; Pratt, R M

    1979-01-01

    In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.

  6. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    PubMed

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

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

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    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. PMID:19468337

  8. 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

    PubMed Central

    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

  9. Multi-physics/scale simulations using particles

    NASA Astrophysics Data System (ADS)

    Koumoutsakos, Petros

    2006-03-01

    Particle simulations of continuum and discrete phenomena can be formulated by following the motion of interacting particles that carry the physical properties of the systems that is being approximated (continuum) or modeled (discrete) by the particles. We identify the common computational characteristics of particle methods and emphasize their key properties that enable the formulation of a novel, systematic framework for multiscale simulations, that can be applicable to the simulation of diverse physical problems. We present novel multiresolution particle methods for continuum (fluid/solid) simulations, using adaptive mesh refinement and wavelets, by relaxing the grid-free character of particle methods and discuss the coupling of scales in continuum-atomistic flow simulations.

  10. Effect of irradiated dib-cAMP on the tonic and phasic activity of human myometrium.

    PubMed

    Schachinger, L; Srivastava, A; Schippel, C; Klöter, H

    1983-06-01

    Cyclic AMP, used as dibutyryl derivative for better permeability, has a relaxing effect on smooth muscle preparations from human uterine tissue (surgical material). The observed decrease of tonus and frequency depends on the concentration applied, shown in the range between 50 and 300 microM. cAMP looses its physiological activity by irradiation in vitro; in addition an inhibitory action of the irradiation products on uterine tissue could be proved. From the data of the Lineweaver-Burk plots, showing the competition between non-irradiated and irradiated cAMP, a ten-to twentyfold higher affinity of the irradiation products to the receptor compared to that of this transmitter could be calculated. The results show that preparations of human origin with beta-receptors behave similarly to animal tissues with beta-receptors. They are further discussed with respect to a better understanding of dose-response curves for chemical and physiological inactivation.

  11. Effect of leaving group on the oligomerization of 5'-AMP on montmorillonite. [Abstract only

    NASA Technical Reports Server (NTRS)

    Prabahar, K. Joseph; Ferris, James P.

    1994-01-01

    The oligomerization of imidazole derivative of 5'-AMP (ImpA) in the presence of montmorillonite clay yields oligomers containing up to 10 monomer units. In these reactions, the heterocyclic base, imidazole is the leaving group. In our present study, we synthesized a series of activated nucleotides of 5'AMP using other leaving groups such as pyrazole, 1,2,4-triazole, piperidine, morpholine, 4-aminopyridine, 4-methylaminopyridine, 4-dimethylaminopyridine, 2-aminobenzimidazole etc. to determine the effect of amine leaving group on the products of the oligomerization reaction. Earlier results from our laboratory showed that the presence AppA in the clay reaction of ImpA enhances the oligomerization reaction to yield higher oligomers. We also studied the effect of AppA in the clay mediated oligomerization reaction of the activated nucleotides. Oligomerization of 2-amino-benzimidazole derivative of 5'-AMP gave higher oligomers containing up to nine monomer units in the presence of AppA.

  12. Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells

    SciTech Connect

    Weston, W.M.; Greene, R.M. )

    1991-01-01

    Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolin-stimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that,in contrast to what has been observed in other systems, ethanol does not affect the production or activity of G{alpha}s in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome.

  13. 8-OH-DPAT facilitated memory consolidation and increased hippocampal and cortical cAMP production.

    PubMed

    Manuel-Apolinar, L; Meneses, A

    2004-01-05

    Animals were submitted to an associative learning task named Pavlovian/instrumental autoshaping (P/I-A) and treated with selective 5-HT1A and 5-HT7 receptor agonists and antagonists. Next, they were sacrificed, their brains removed, dissected and changes on cortical and hippocampal cyclic adenosine monophosphate (cAMP) production were determined. Results revealed that, the 8-OH-DPAT treatment facilitated memory consolidation of autoshaping and that effect was blocked completely by WAY100635 and partially by DR4004. WAY100635 or DR4004 alone had no effect on autoshaping. The cAMP results were complex and yielded no clear relationship to the memory results. Thus, cortical and hippocampal increased on cAMP production was observed following administration of the 5-HT(1A/7) agonist 8-OH-DPAT. The memory effect was, completely or partially, reversed by the selective antagonists WAY100635 (5-HT1A) or DR4004 (5-HT7), respectively.

  14. Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange Mass Spectrometry

    PubMed Central

    Landgraf, Rachelle R.; Goswami, Devrishi; Rajamohan, Francis; Harris, Melissa S.; Calabrese, Matthew; Hoth, Lise R.; Magyar, Rachelle; Pascal, Bruce D.; Chalmers, Michael J.; Busby, Scott A.; Kurumbail, Ravi; Griffin, Patrick R.

    2013-01-01

    Summary AMP-Activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme by hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit suggesting that the molecular binding site of latter resides between the α and β subunits. The distinct short and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands. PMID:24076403

  15. Cyclic AMP-dependent phosphorylation of neuronal nitric oxide synthase mediates penile erection.

    PubMed

    Hurt, K Joseph; Sezen, Sena F; Lagoda, Gwen F; Musicki, Biljana; Rameau, Gerald A; Snyder, Solomon H; Burnett, Arthur L

    2012-10-09

    Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor L-NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction.

  16. The cyclic AMP signaling pathway: Exploring targets for successful drug discovery (Review)

    PubMed Central

    YAN, KUO; GAO, LI-NA; CUI, YUAN-LU; ZHANG, YI; ZHOU, XIN

    2016-01-01

    During development of disease, complex intracellular signaling pathways regulate an intricate series of events, including resistance to external toxins, the secretion of cytokines and the production of pathological phenomena. Adenosine 3′,5′-cyclic monophosphate (cAMP) is a nucleotide that acts as a key second messenger in numerous signal transduction pathways. cAMP regulates various cellular functions, including cell growth and differentiation, gene transcription and protein expression. This review aimed to provide an understanding of the effects of the cAMP signaling pathway and the associated factors on disease occurrence and development by examining the information from a new perspective. These novel insights aimed to promote the development of novel therapeutic approaches and aid in the development of new drugs. PMID:27035868

  17. A reduced mechanical model for cAMP-modulated gating in HCN channels

    PubMed Central

    Weißgraeber, Stephanie; Saponaro, Andrea; Thiel, Gerhard; Hamacher, Kay

    2017-01-01

    We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP. PMID:28074902

  18. Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

    NASA Technical Reports Server (NTRS)

    Fong, J. H.; Ingber, D. E.

    1996-01-01

    We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

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

    PubMed

    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.

  20. A reduced mechanical model for cAMP-modulated gating in HCN channels

    NASA Astrophysics Data System (ADS)

    Weißgraeber, Stephanie; Saponaro, Andrea; Thiel, Gerhard; Hamacher, Kay

    2017-01-01

    We developed an in silico mechanical model to analyze the process of cAMP-induced conformational modulations in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which conduct cations across the membrane of mammalian heart and brain cells. The structural analysis reveals a quaternary twist in the cytosolic parts of the four subunits in the channel tetramer. This motion augments the intrinsic dynamics of the very same protein structure. The pronounced differences between the cAMP bound and unbound form include a mutual interaction between the C-linker of the cyclic nucleotide binding domain (CNBD) and the linker between the S4 and S5 transmembrane domain of the channel. This allows a mechanistic annotation of the twisting motion in relation to the allosteric modulation of voltage-dependent gating of this channel by cAMP.

  1. Advanced Science.

    ERIC Educational Resources Information Center

    Coles, Mike; Nelms, Rick

    1996-01-01

    Describes a study that explores the depth and breadth of scientific facts, principles, and procedures which are required in the Advanced General National Vocational Qualifications (GNVQ) science through comparison with GCE Advanced level. The final report takes account of the updated 1996 version of GNVQ science. (DDR)

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

    PubMed Central

    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

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

    PubMed

    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.

  4. Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity.

    PubMed

    Guinzberg, Raquel; Díaz-Cruz, Antonio; Acosta-Trujillo, Carlos; Vilchis-Landeros, María Magdalena; Vázquez-Meza, Héctor; Lozano-Flores, Carlos; Chiquete-Felix, Natalia; Varela-Echavarría, Alfredo; Uribe-Carvajal, Salvador; Riveros-Rosas, Héctor; Piña, Enrique

    2017-01-01

    Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

  5. PET measurements of cAMP-mediated phosphodiesterase-4 with (R)-[11C]rolipram.

    PubMed

    Kenk, Miran; Thomas, Adam; Lortie, Mireille; Dekemp, Rob; Beanlands, Rob S; Dasilva, Jean N

    2011-01-01

    Cyclic adenosine monophosphate (cAMP) is the common second messenger in signal-transduction cascades originating at a number of monoamine receptors involved in neurotransmission, cardiac function and smooth muscle contraction. Altered regulation of cAMP synthesis (at receptors, G-protein subunits or adenylyl cyclase) and breakdown by phosphodiesterase (PDE) enzymes have been implicated in a number of pathologies. The PDE4 inhibitor (R)-rolipram, and the less active (S)- enantiomer, have been labeled with carbon-11 and characterized by in vivo and in vitro experiments for use in the evaluation of altered PDE4 levels in the brain and cardiac tissues. (R)-[11C]Rolipram has been shown to bind selectively to PDE4 over other PDE isozymes, with specific binding reflecting approximately 80 and 40% of the total detected radioactivity in the rat brain and the heart, respectively. Tracer retention in PDE4-rich tissues is increased by cAMP-elevating treatments, as detected by in vivo PET studies and ex vivo biodistribution experiments. In vivo PET imaging studies display strong region-specific signal in the brain and heart, as evaluated in rats, pigs, monkeys and humans. Impaired cAMP-mediated signaling was observed in animal models of aging, obesity, anthracycline-induced cardiotoxicity and myocardial infarction using (R)-[11C]rolipram. Given the critical role of cAMP in multiple hormonal pathways, the good safety profile and well-characterized pharmacokinetics, (R)-[11C]rolipram PET imaging provides a novel tool for serial monitoring of cAMP-mediated signaling at the PDE4 level, yielding insight into pathological progression with potential for directing therapy.

  6. -Adrenergic receptors on rat ventricular myocytes: characteristics and linkage to cAMP metabolism

    SciTech Connect

    Buxton, I.L.O.; Brunton, L.L.

    1986-08-01

    When incubated with purified cardiomyocytes from adult rat ventricle, the 1-antagonist (TH)prazosin binds to a single class of sites with high affinity. Competition for (TH)prazosin binding by the 2-selective antagonist yohimbine and the nonselective -antagonist phentolamine demonstrates that these receptors are of the 1-subtype. In addition, incubation of myocyte membranes with (TH)yohimbine results in no measurable specific binding. Agonist competition for (TH)prazosin binding to membranes prepared from purified myocytes demonstrates the presence of two components of binding: 28% of 1-receptors interact with norepinephrine with high affinity (K/sub D/ = 36 nM), whereas the majority of receptors (72%) have a low affinity for agonist (K/sub D/ = 2.2 M). After addition of 10 M GTP, norepinephrine competes for (TH)prazosin binding to a single class of sites with lower affinity (K/sub D/ = 2.2 M). Incubation of intact myocytes for 2 min with 1 M norepinephrine leads to significantly less cyclic AMP (cAMP) accumulation than stimulation with either norepinephrine plus prazosin or isoproterenol. Likewise, incubation of intact myocytes with 10 W M norepinephrine leads to significantly less activation of cAMP-dependent protein kinase than when myocytes are stimulated by both norepinephrine and the 1-adrenergic antagonist, prazosin or the US -adrenergic agonist, isoproterenol. They conclude that the cardiomyocyte 1 receptor is coupled to a guanine nucleotide-binding protein, that 1-receptors are functionally linked to decreased intracellular cAMP content, and that this change in cellular cAMP is expressed as described activation of cAMP-dependent protein kinase.

  7. The role of cAMP-mediated intracellular signaling in regulating Na+ uptake in zebrafish larvae

    PubMed Central

    Kumai, Yusuke; Kwong, Raymond W. M.

    2013-01-01

    In the current study, the role of cAMP in stimulating Na+ uptake in larval zebrafish was investigated. Treating larvae at 4 days postfertilization (dpf) with 10 μM forskolin or 1 μM 8-bromo cAMP significantly increased Na+ uptake by three-fold and twofold, respectively. The cAMP-dependent stimulation of Na+ uptake was probably unrelated to protein trafficking via microtubules because pretreatment with 200 μM colchicine or 30 μM nocodazole did not attenuate the magnitude of the response. Na+ uptake was stimulated markedly following acute (2 h) exposure to acidic water. The acid-induced increase in Na+ uptake was accompanied by a twofold elevation in whole body cAMP levels and attenuated by inhibiting PKA with 10 μM H-89. Knockdown of Na+-H+ exchanger 3b (NHE3b) attenuated, but did not abolish, the stimulation of Na+ uptake during forskolin treatment. In glial cell missing 2 morphants, in which the role of NHE3b in Na+ uptake is diminished and the Na+-Cl− cotransporter (NCC) becomes the predominant route of Na+ entry, forskolin treatment continued to increase Na+ uptake. These data suggest that at least NHE3b and NCC are targeted by cAMP in zebrafish larvae. Staining of larvae with fluorescent forskolin and propranolol revealed the presence of transmembrane adenylyl cyclase within multiple subtypes of ionocytes expressing β-adrenergic receptors. Taken together, results of the present study demonstrate that cAMP-mediated intracellular signaling may regulate multiple Na+ transporters and plays an important role in regulating Na+ uptake in zebrafish larvae during acute exposure to an acidic environment. PMID:24259461

  8. Histamine-stimulated cyclic AMP formation in the chick pineal gland: role of protein kinase C.

    PubMed

    Zawilska, J B; Woldan-Tambor, A; Nowak, J Z

    1997-08-15

    The role of protein kinase C (PKC) in histamine (HA)-stimulated cyclic AMP formation in intact chick pineal glands was investigated. In the pineal gland of chick HA, 2-methylHA, 4-methylHA, and N alpha, N alpha-dimethylHA potently increased cyclic AMP accumulation in a concentration-dependent manner. Treatment of intact glands with PKC inhibitors, i.e. chelerythrine and stautosporine, reduced the stimulatory effect of the HA-ergic compounds on cyclic AMP formation. HA, 2-methylHA, 4-methylHA, and N alpha, N alpha-dimethylHA significantly increased inositol-1,4,5-trisphosphate (IP3) levels in intact chick pineal glands, indicating their activities on phospholipase C and 1,2-diacylglycerol formation. The stimulatory effect of HA on IP3 levels was antagonized by aminopotentidine, a potent blocker of H2-like HA receptors in avian pineal gland. Preincubation of chick pineal glands with a PKC activator, 4 beta-phorbol 12, 13-dibutyrate (4 beta-PDB), enhanced the accumulation of cyclic AMP elicited by HA, 2-methylHA, 4-methylHA, and N alpha, N alpha-dimethylHA. On the other hand, 4 beta-phorbol, inactive on the PKC, was ineffective. Our results point to the possibility that PKC is involved in the regulation by HA of cyclic AMP synthesis in the pineal gland of chick. Furthermore, the cyclic AMP response to pineal HA receptor stimulation can be positively modulated by a concomitant activation of the PKC pathway.

  9. Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling

    PubMed Central

    Zhang, Chun-Lei; Moshous, Despina; Studer, Vera; Schneider, Jacques; Genoud, Christel; Fossoud, Catherine; Gambino, Frédéric; Khelfaoui, Malik; Müller, Christian; Bartholdi, Deborah; Rossez, Helene; Stiess, Michael; Houbaert, Xander; Jaussi, Rolf; Frey, Daniel; Kammerer, Richard A.; Deupi, Xavier; de Villartay, Jean-Pierre; Lüthi, Andreas; Humeau, Yann; Pieters, Jean

    2014-01-01

    Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic–AMP–protein kinase A–dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1–deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes. PMID:24667537

  10. Diverse effects of cyclic AMP variants on osteogenic and adipogenic differentiation of human mesenchymal stromal cells.

    PubMed

    Doorn, Joyce; Leusink, Maarten; Groen, Nathalie; van de Peppel, Jeroen; van Leeuwen, Johannes P T M; van Blitterswijk, Clemens A; de Boer, Jan

    2012-07-01

    Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) may potentially be used in cell-based bone tissue-engineering applications to enhance the bone-forming potential of these cells. Osteogenic differentiation and adipogenic differentiation are thought to be mutually exclusive, and although several signaling pathways and cues that induce osteogenic or adipogenic differentiation, respectively, have been identified, there is no general consensus on how to optimally differentiate hMSCs into the osteogenic lineage. Some pathways have also been reported to be involved in both adipogenic and osteogenic differentiation, as for example, the protein kinase A (PKA) pathway, and the aim of this study was to investigate the role of cAMP/PKA signaling in differentiation of hMSCs in more detail. We show that activation of this pathway with dibutyryl-cAMP results in enhanced alkaline phosphatase expression, whereas another cAMP analog induces adipogenesis in long-term mineralization cultures. Adipogenic differentiation, induced by 8-bromo-cAMP, was accompanied by stronger PKA activity and higher expression of cAMP-responsive genes, suggesting that stronger activation correlates with adipogenic differentiation. In addition, a whole-genome expression analysis showed an increase in expression of adipogenic genes in 8-br-cAMP-treated cells. Furthermore, by means of quantitative polymerase chain reaction, we show differences in peroxisome proliferator-activated receptor-γ activation, either alone or in combination with dexamethasone, thus demonstrating differential effects of the PKA pathway, most likely depending on its mode of activation.

  11. Phosphodiesterases in the rat ovary: effect of cAMP in primordial follicles.

    PubMed

    Petersen, Tonny Studsgaard; Stahlhut, Martin; Andersen, Claus Yding

    2015-07-01

    Phosphodiesterases (PDEs) are important regulators of the intracellular cAMP concentration, which is a central second messenger that affects a multitude of intracellular functions. In the ovaries, cAMP exerts diverse functions, including regulation of ovulation and it has been suggested that augmented cAMP levels stimulate primordial follicle growth. The present study examined the gene expression, enzyme activity and immunolocalization of the different cAMP hydrolysing PDEs families in the rat ovary. Further, the effect of PDE4 inhibition on primordial follicle activation in cultured neonatal rat ovaries was also evaluated. We found varied expression of all eight families in the ovary with Pde7b and Pde8a having the highest expression each accounting for more than 20% of the total PDE mRNA. PDE4 accounted for 15-26% of the total PDE activity. Immunoreactive PDE11A was found in the oocytes and PDE2A in the corpora lutea. Incubating neonatal rat ovaries with PDE4 inhibitors did not increase primordial follicle activation or change the expression of the developing follicle markers Gdf9, Amh, Inha, the proliferation marker Mki67 or the primordial follicle marker Tmeff2. In addition, the cAMP analogue 8-bromo-cAMP did not increase AKT1 or FOXO3A phosphorylation associated with follicle activation or increase the expression of Kitlg known to be associated with follicle differentiation but did increase the Tmeff2, Mki67 and Inha expression in a dose-dependent manner. In conclusion, this study shows that both Pde7b and Pde8a are highly expressed in the rodent ovary and that PDE4 inhibition does not cause an increase in primordial follicle activation.

  12. Anti-tumor activity and mechanism of action for a cyanoaziridine-derivative, AMP423

    PubMed Central

    Wisner, Lee; Samulitis, Betty K.; Landowski, Terry H.; Remers, William A.

    2012-01-01

    Purpose Preclinical studies evaluated the anti-tumor activity and mechanism of action of AMP423, a naphthyl derivative of 2-cyanoaziridine-1-carboxamide with structural similarity to the pro-oxidant anti-tumor agent imexon. Methods The cytotoxic potency was evaluated in vitro against a variety of human cancer cell lines. Mechanism-of-action studies were performed in the human 8226/S myeloma cell line and its imexon-resistant variant, 8226/IM10. In vivo activity was evaluated against human myeloma and lymphoma xenografts in SCID mice. Pharmacokinetics and toxicology were investigated in non-tumor-bearing mice. Results The 72-h IC50s for all cell types ranged from 2 to 36 μM, across a wide variety of human cancer cell lines. AMP423 was active in SCID mice bearing 8226/S myeloma and SU-DHL-6 B-cell lymphoma tumors, with a median tumor growth delay (T–C) of 21 days (P = 0.0002) and 5 days (P = 0.004), respectively, and a median tumor growth inhibition (T/C) of 33.3% (P = 0.03) and 82% (P = 0.01), respectively. In non-tumor-bearing mice, AMP423 was not myelosuppressive. Mechanistic studies show that AMP423’s mode of cell death is a mixture of necrosis and apoptosis, with generation of reactive oxygen species, inhibition of protein synthesis, and a decrease in reduced sulfhydryl levels, but no alkylation of nucleophiles. Unlike its structural analog imexon, which causes cell cycle arrest in G2/M, AMP423 induces the accumulation of cells in S-phase. Conclusions AMP423 has pro-oxidant effects similar to imexon, has greater cytotoxic potency in vitro, and has anti-tumor activity in hematologic tumors in vivo. PMID:22186884

  13. Cyclic AMP stimulates neurite outgrowth of lamprey reticulospinal neurons without substantially altering their biophysical properties.

    PubMed

    Pale, T; Frisch, E B; McClellan, A D

    2013-08-15

    Reticulospinal (RS) neurons are critical for initiation of locomotor behavior, and following spinal cord injury (SCI) in the lamprey, the axons of these neurons regenerate and restore locomotor behavior within a few weeks. For lamprey RS neurons in culture, experimental induction of calcium influx, either in the growth cone or cell body, is inhibitory for neurite outgrowth. Following SCI, these neurons partially downregulate calcium channel expression, which would be expected to reduce calcium influx and possibly provide supportive conditions for axonal regeneration. In the present study, it was tested whether activation of second messenger signaling pathways stimulates neurite outgrowth of lamprey RS neurons without altering their electrical properties (e.g. spike broadening) so as to possibly increase calcium influx and compromise axonal growth. First, activation of cAMP pathways with forskolin or dbcAMP stimulated neurite outgrowth of RS neurons in culture in a PKA-dependent manner, while activation of cGMP signaling pathways with dbcGMP inhibited outgrowth. Second, neurophysiological recordings from uninjured RS neurons in isolated lamprey brain-spinal cord preparations indicated that dbcAMP or dbcGMP did not significantly affect any of the measured electrical properties. In contrast, for uninjured RS neurons, forskolin increased action potential duration, which might have increased calcium influx, but did not significantly affect most other electrical properties. Importantly, for injured RS neurons during the period of axonal regeneration, forskolin did not significantly alter their electrical properties. Taken together, these results suggest that activation of cAMP signaling by dbcAMP stimulates neurite outgrowth, but does not alter the electrical properties of lamprey RS neurons in such a way that would be expected to induce calcium influx. In conclusion, our results suggest that activation of cAMP pathways alone, without compensation for possible

  14. Isolation of novel ribozymes that ligate AMP-activated RNA substrates

    NASA Technical Reports Server (NTRS)

    Hager, A. J.; Szostak, J. W.

    1997-01-01

    BACKGROUND: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated. RESULTS: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site. CONCLUSIONS: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.

  15. Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

    SciTech Connect

    X Xu; Z Vysotskaya; Q Liu; L Zhou

    2011-12-31

    Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 {angstrom}. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a {approx}3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between {beta}4 and {beta}5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.

  16. AIP inactivation leads to pituitary tumorigenesis through defective Gαi-cAMP signaling.

    PubMed

    Tuominen, I; Heliövaara, E; Raitila, A; Rautiainen, M-R; Mehine, M; Katainen, R; Donner, I; Aittomäki, V; Lehtonen, H J; Ahlsten, M; Kivipelto, L; Schalin-Jäntti, C; Arola, J; Hautaniemi, S; Karhu, A

    2015-02-26

    The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

  17. Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events.

    PubMed Central

    Dowd, D R; Miesfeld, R L

    1992-01-01

    WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways. Images PMID:1378529

  18. PD98059 and U0126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway.

    PubMed

    Dokladda, Kanchana; Green, Kevin A; Pan, David A; Hardie, D Grahame

    2005-01-03

    The MAP kinase pathway inhibitor U0126 caused phosphorylation and activation of AMP-activated protein kinase (AMPK) and increased phosphorylation of its downstream target acetyl-CoA carboxylase, in HEK293 cells. This effect only occurred in cells expressing the upstream kinase, LKB1. Of two other widely used MAP kinase pathway inhibitors not closely related in structure to U0126, PD98059 also activated AMPK but PD184352 did not. U0126 and PD98059, but not PD184352, also increased the cellular ADP:ATP and AMP:ATP ratios, accounting for their ability to activate AMPK. These results suggest the need for caution in interpreting experiments conducted using U0126 and PD98059.

  19. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

    PubMed Central

    Peakman, M. C.; Hill, S. J.

    1994-01-01

    1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B

  20. Biophysical techniques for detection of cAMP and cGMP in living cells.

    PubMed

    Sprenger, Julia U; Nikolaev, Viacheslav O

    2013-04-12

    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.

  1. AMP-activated protein kinase—an energy sensor that regulates all aspects of cell function

    PubMed Central

    Hardie, D. Grahame

    2011-01-01

    AMP-activated protein kinase (AMPK) is a sensor of energy status that maintains cellular energy homeostasis. It arose very early during eukaryotic evolution, and its ancestral role may have been in the response to starvation. Recent work shows that the kinase is activated by increases not only in AMP, but also in ADP. Although best known for its effects on metabolism, AMPK has many other functions, including regulation of mitochondrial biogenesis and disposal, autophagy, cell polarity, and cell growth and proliferation. Both tumor cells and viruses establish mechanisms to down-regulate AMPK, allowing them to escape its restraining influences on growth. PMID:21937710

  2. [Role of antimicrobial peptides (AMP) and pattern recognition receptors (PRR) in the intestinal mucosa homeostasis].

    PubMed

    Lapis, Károly

    2009-11-22

    Homeostasis and integrity of bowel mucosa is assured by well controlled mechanical, biochemical and immunological mechanisms. First line of defense is presented by the antimicrobial peptides (AMP), which form a continuous layer on the bowel surface, produced by intestinal specific (Paneth) and non-specific epithelial cells. AMPs have a significant antimicrobial, antifungal and antiviral, as well as immunomodulatory effects. Next line of defense is the pattern recognition receptors (PRR), which allows identifying conservative molecular patterns of different pathogens, and starts antimicrobial and inflammatory mechanisms through gene-expression induction. We review the most recent knowledge and studies concerning these mechanisms.

  3. Transmission of ESBL/AmpC-producing Escherichia coli from broiler chicken farms to surrounding areas.

    PubMed

    Laube, H; Friese, A; von Salviati, C; Guerra, B; Rösler, U

    2014-08-27

    Although previous studies have demonstrated high carriage of ESBL/AmpC-producing Escherichia coli in livestock, especially in broiler chickens, data on emission sources of these bacteria into the environment are still rare. Therefore, this study was designed to systematically investigate the occurrence of ESBL/AmpC-producing E. coli in slurry, air (inside animal houses), ambient air (outside animal houses) and on soil surfaces in the areas surrounding of seven ESBL/AmpC-positive broiler chicken fattening farms, including investigation of the possible spread of these bacteria via the faecal route and/or exhaust air into the environment. Seven German broiler fattening farms were each investigated at three points in time (3-36 h after restocking, 14-18 and 26-35 days after housing) during one fattening period. The occurrence of ESBL/AmpC genes in the investigated samples was confirmed by PCR, detecting blaCTX-M, blaSHV, blaTEM and blaCMY-genes, and, if necessary, by sequencing and/or the disc diffusion method. The results showed a wide spread of ESBL/AmpC-producing E. coli in broiler farms, as well as emissions into the surroundings. 12 out of 14 (86%) slurry samples were positive for ESBL/AmpC-producing E. coli. Additionally, 28.8% (n=23/80) of boot swabs taken from various surfaces in the areas surrounding of the farms as well as 7.5% (n=3/40) of the exhaust air samples turned out to be positive for these microorganisms. Moreover, a small proportion of air samples from inside the barns were ESBL/AmpC-positive. By comparing selected isolates using pulsed field gel electrophoresis, we proved that faecal and airborne transfer of ESBL/AmpC-producing microorganisms from broiler fattening farms to the surrounding areas is possible. Two isolates from farm G2 (slurry and boot swab 50 m downwind), two isolates from farm G3 (slurry and individual animal swab) as well as two isolates from farm G6 (air sample in the barn and air sample 50 m downwind) showed 100% similarity in

  4. C-terminal dimerization of apo-cyclic AMP receptor protein validated in solution.

    PubMed

    Sim, Dae-Won; Choi, Jae Wan; Kim, Ji-Hun; Ryu, Kyoung-Seok; Kim, Myeongkyu; Yu, Hee-Wan; Jo, Ku-Sung; Kim, Eun-Hee; Seo, Min-Duk; Jeon, Young Ho; Lee, Bong-Jin; Kim, Young Pil; Won, Hyung-Sik

    2017-04-01

    Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo-CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP.

  5. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    SciTech Connect

    Hwang, Tsong-Long; Tang, Ming-Chi; Kuo, Liang-Mou; Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching

    2012-04-15

    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  6. Catalytic roles of the AMP at the 3' end of tRNAs

    NASA Technical Reports Server (NTRS)

    Wickramasinghe, N. S.; Lacey, J. C. Jr; Lacey JC, J. r. (Principal Investigator)

    1994-01-01

    Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3' terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3' terminus of tRNA.

  7. Rolipram stimulates angiogenesis and attenuates neuronal apoptosis through the cAMP/cAMP-responsive element binding protein pathway following ischemic stroke in rats.

    PubMed

    Hu, Shouye; Cao, Qingwen; Xu, Peng; Ji, Wenchen; Wang, Gang; Zhang, Yuelin

    2016-03-01

    Rolipram, a phosphodiesterase-4 inhibitor, can activate the cyclic adenosine monophosphate (cAMP)/cAMP-responsive element binding protein (CREB) pathway to facilitate functional recovery following ischemic stroke. However, to date, the effects of rolipram on angiogenesis and cerebral ischemia-induced neuronal apoptosis are yet to be fully elucidated. In this study, the aim was to reveal the effect of rolipram on the angiogenesis and neuronal apoptosis following brain cerebral ischemia. Rat models of ischemic stroke were established following transient middle cerebral artery occlusion and rolipram was administered for three, seven and 14 days. The results were examined using behavioral tests, triphenyl tetrazolium chloride staining, immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to evaluate the effects of rolipram therapy on functional outcome, angiogenesis and apoptosis. Western blot analysis was used to show the phosphorylated- (p-)CREB protein level in the ischemic hemisphere. The rolipram treatment group exhibited a marked reduction in infarct size and modified neurological severity score compared with the vehicle group, and rolipram treatment significantly promoted the microvessel density in the ischemic boundary region and increased p-CREB protein levels in the ischemic hemisphere. Furthermore, a significant reduction in the number of TUNEL-positive cells was observed in the rolipram group compared with the vehicle group. These findings suggest that rolipram has the ability to attenuate cerebral ischemic injury, stimulate angiogenesis and reduce neuronal apoptosis though the cAMP/CREB pathway.

  8. Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2009-09-02

    The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/{Delta}598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions ({le}1 mg ml{sup -1}), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of {approx}10 mg ml{sup -1}. Initial crystals were obtained by the microbatch method in the presence of a U{sub 10} RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/{Delta}598-664 in complex with AMP-PNP and U{sub 10} belonged to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 {angstrom}, and diffracted X-rays to beyond 1.9 {angstrom} resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.

  9. Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function.

    PubMed

    Saponaro, Andrea; Pauleta, Sofia R; Cantini, Francesca; Matzapetakis, Manolis; Hammann, Christian; Donadoni, Chiara; Hu, Lei; Thiel, Gerhard; Banci, Lucia; Santoro, Bina; Moroni, Anna

    2014-10-07

    cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

  10. cAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells.

    PubMed

    Zhang, Linxia; Seitz, Linsey C; Abramczyk, Amy M; Liu, Li; Chan, Christina

    2011-03-01

    The intracellular second messenger cAMP is frequently used in induction media to induce mesenchymal stem cells (MSCs) into neural lineage cells. To date, an understanding of the role cAMP exerts on MSCs and whether cAMP can induce MSCs into functional neurons is still lacking. We found cAMP initiated neuron-like morphology changes early and neural differentiation much later. The early phase changes in morphology were due to cell shrinkage, which subsequently rendered some cells apoptotic. While the morphology changes occurred prior to the expression of neural markers, it is not required for neural marker expression and the two processes are differentially regulated downstream of cAMP-activated protein kinase A. cAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate, and potassium chloride. However, only some of the cells induced by cAMP responded to the three neuronal activators and further lack the neuronal morphology, suggesting that although cAMP is able to direct MSCs towards neural differentiation, they do not achieve terminal differentiation.

  11. Termination of cAMP signals by Ca2+ and Gαi via extracellular Ca2+ sensors

    PubMed Central

    Gerbino, Andrea; Ruder, Warren C.; Curci, Silvana; Pozzan, Tullio; Zaccolo, Manuela; Hofer, Aldebaran M.

    2005-01-01

    Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca2+-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer–dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin–sensitive Gαi and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca2+-mobilizing agonists under physiological conditions. PMID:16247029

  12. 78 FR 1252 - CalAmp Wireless Networks Corporation (CWNC), Satellite Products Division, Including On-Site...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-08

    ... Division, Including On-Site Leased Workers From Select Staffing, Oxnard, CA; CalAmp Wireless Networks... Select Staffing, Oxnard, California (TA-W-80,399). The workers are engaged in the production of converter... Select Staffing, Oxnard, California (TA-W-80,399) and CalAmp Wireless Networks Corporation...

  13. cAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells

    PubMed Central

    Zhang, Linxia; Seitz, Linsey C.; Abramczyk, Amy M.; Liu, Li

    2010-01-01

    The intracellular second messenger cAMP is frequently used in induction media to induce mesenchymal stem cells (MSCs) into neural lineage cells. To date, an understanding of the role cAMP exerts on MSCs and whether cAMP can induce MSCs into functional neurons is still lacking. We found cAMP initiated neuron-like morphology changes early and neural differentiation much later. The early phase changes in morphology were due to cell shrinkage, which subsequently rendered some cells apoptotic. While the morphology changes occurred prior to the expression of neural markers, it is not required for neural marker expression and the two processes are differentially regulated downstream of cAMP-activated protein kinase A. cAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate, and potassium chloride. However, only some of the cells induced by cAMP responded to the three neuronal activators and further lack the neuronal morphology, suggesting that although cAMP is able to direct MSCs towards neural differentiation, they do not achieve terminal differentiation. PMID:20725762

  14. cAMP controls the balance of the propulsive forces generated by the two flagella of Chlamydomonas.

    PubMed

    Saegusa, Yu; Yoshimura, Kenjiro

    2015-08-01

    The motility of cilia and flagella of eukaryotic cells is controlled by second messengers such as Ca(2+), cAMP, and cGMP. In this study, the cAMP-dependent control of flagellar bending of Chlamydomonas is investigated by applying cAMP through photolysis of 4,5-dimethoxy-2-nitrobenzyl adenosine 3',5'-cyclicmonophosphate (caged cAMP). When cAMP is applied to demembranated and reactivated cells, cells begin to swim with a larger helical path. This change is due to a larger turn about the axis normal to the anterior-posterior axis, indicating an increased imbalance in the propulsive forces generated by the cis-flagellum (flagellum nearer to the eyespot) and trans-flagellum (flagellum farther from the eyespot). Consistently, when cAMP is applied to isolated axonemes, some axonemes show attenuated motility whereas others do not. Axonemes from uni1 mutants, which have only trans-flagella, do not respond to cAMP. These observations indicate that cAMP controls the balance of the forces generated by cis- and trans-flagella in Chlamydomonas.

  15. Synergistic induction of insulin resistance by endothelin-1 and cAMP in 3T3-L1 adipocytes.

    PubMed

    Chai, Shin-Pei; Fong, Jim C

    2015-10-01

    Both endothelin-1 (ET-1) and cAMP are implicated for inducing insulin resistance. Since we have shown previously that there is a crosstalk between ET-1 and cAMP signaling pathways in regulating glucose uptake in 3T3-L1 adipocytes, we extended our investigation in this study on whether they may have a synergistic effect on inducing insulin resistance. Our results showed that it was indeed the case. Insulin-stimulated glucose uptake, phosphorylation of PKB, IRS-1-associated PI3K, and IRS-1 tyrosine phosphorylation were all inhibited by ET-1 and 8-bromo cAMP in a synergistic manner. IRS-1 protein levels were similarly decreased by ET-1 and 8-bromo cAMP, attributable to suppressed mRNA expression. In addition, after correction for the loss in IRS-1 protein, the inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation or IRS-1-associated PI3K was mainly caused by cAMP. Moreover, whereas IRS-2 protein levels were increased by cAMP or ET-1 plus cAMP, insulin-stimulated IRS-2-associated PI3K activities were abolished by both treatments. Furthermore, ET-1 and β-adrenergic agonists had similar synergistic inhibition on insulin-stimulated glucose uptake. In conclusion, we have shown that ET-1 and cAMP may synergistically induce insulin resistance in adipocytes via inhibiting IRS-1 expression as well as insulin-stimulated IRS-1/IRS-2 activities.

  16. Extended-spectrum β-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in animals: a threat for humans?

    PubMed

    Madec, J-Y; Haenni, M; Nordmann, P; Poirel, L

    2017-01-29

    There has been a great and long-term concern that extended-spectrum β-lactamase (ESBL)/AmpC- and carbapenemase-producing Enterobacteriaceae occurring in animals may constitute a public-health issue. A large number of factors with complex interrelations contribute to the spread of those bacteria among animals and humans. ESBL/AmpC- or carbapenemase-encoding genes are most often located on mobile genetic elements favouring their dissemination. Some shared reservoirs of ESBL/AmpC or carbapenemase genes, plasmids or clones have been identified and suggest cross-transmissions. Even though exposure to animals is regarded as a risk factor, evidence for a direct transfer of ESBL/AmpC-producing bacteria from animals to humans through close contacts is limited. Nonetheless, the size of the commensal ESBL/AmpC reservoir in non-human sources is dramatically rising. This may constitute an indirect risk to public health by increasing the gene pool from which pathogenic bacteria can pick up ESBL/AmpC/carbapenemase genes. The extent to which food contributes to potential transmission of ESBL/AmpC producers to humans is also not well established. Overall, events leading to the occurrence of ESBL/AmpC- and carbapenemase-encoding genes in animals seem very much multifactorial. The impact of animal reservoirs on human health still remains debatable and unclear; nonetheless, there are some examples of direct links that have been identified.

  17. Expression and activity of the 5'-AMP-activated protein kinase pathway in selected tissues during chicken embryonic development.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 5’-AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase and a key part of a kinase signaling cascade that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating m...

  18. Cyclic AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain cells

    SciTech Connect

    Rossie, S.; Catterall, W.A.

    1986-03-05

    The ..cap alpha.. subunit of the voltage-sensitive Na channel from rat brain is phosphorylated by cAMP-dependent protein kinase in purified preparations and in synaptosomes. The authors have begun to study cAMP-dependent phosphorylation of Na channels in intact cells. Rat brain cells collected at embryonic day 15 and maintained in culture for approximately 21 days were subjected to treatments designed to increase intracellular cAMP. Cells were solubilized and Na channels were isolated by immunoprecipitation, then rephosphorylated with the catalytic subunit of cAMP-dependent protein kinase and /sup 32/P-ATP, to allow incorporation of /sup 32/P into available cAMP-dependent phosphorylation sites of Na channels. The amount of /sup 32/P incorporated into channel is inversely proportional to the extent of endogenous phosphorylation. Treatment of cells with forskolin inhibited rephosphorylation of Na channels, indicating that enhanced endogenous phosphorylation of channels had occurred. The effect of forskolin on cell surface Na channels occurred rapidly, was sustained over 30 min., and was half-maximal at 6..mu..M. 8-Br-cAMP, (EC/sub 50/-5mM) and isobutylmethylxanthine (EC/sub 50/-60..mu..M) also caused inhibition of /sup 32/P incorporation into Na channels. These results indicate that the extent of cAMP-dependent phosphorylation of voltage-sensitive Na channels in intact brain neurons is modified by changes in intracellular levels of cAMP.

  19. Structural Insights into the Mechanism of the Allosteric Transitions of Mycobacterium tuberculosis cAMP Receptor Protein

    SciTech Connect

    Reddy, Manchi C.M.; Palaninathan, Satheesh K.; Bruning, John B.; Thurman, Cory; Smith, Danielle; Sacchettini, James C.

    2010-02-11

    The cAMP receptor protein (CRP) from Mycobacterium tuberculosis is a cAMP-responsive global transcriptional regulator, responsible for the regulation of a multitude of diverse proteins. We have determined the crystal structures of the CRP {center_dot} cAMP and CRP {center_dot} N{sup 6}-cAMP derivative-bound forms of the enzyme to 2.2- and 2.3 {angstrom}-resolution, respectively, to investigate cAMP-mediated conformational and structural changes. The allosteric switch from the open, inactive conformation to the closed, active conformation begins with a number of changes in the ligand-binding cavity upon cAMP binding. These subtle structural changes and numerous non-bonding interactions between cAMP, the N-domain residues, and the C-domain helices demonstrate that the N-domain hairpin loop acts as a structural mediator of the allosteric switch. Based on the CRP {center_dot} N{sup 6}-cAMP crystal structure, binding of N{sup 6}-cAMP with a bulkier methylphenylethyl extension from the N{sup 6} atom stabilizes the cAMP-binding domain, N-domain hairpin, and C-terminal domain in a similar manner as that of the CRP {center_dot} cAMP structure, maintaining structural integrity within the subunits. However, the bulkier N{sup 6} extension of N{sup 6}-cAMP (in R conformation) is accommodated only in subunit A with minor changes, whereas in subunit B, the N{sup 6} extension is in the S conformation hindering the hinge region of the central helix. As a result, the entire N-domain and the C-domain of subunit B integrated by the cAMP portion of this ligand, together tilt away ({approx}7{sup o} tilt) from central helix C, positioning the helix-turn-helix motif in an unfavorable position for the DNA substrate, asymmetrically. Together, these crystal structures demonstrate the mechanism of action of the cAMP molecule and its role in integrating the active CRP structure.

  20. Glucose- and Hormone-Induced cAMP Oscillations in α- and β-Cells Within Intact Pancreatic Islets

    PubMed Central

    Tian, Geng; Sandler, Stellan; Gylfe, Erik; Tengholm, Anders

    2011-01-01

    OBJECTIVE cAMP is a critical messenger for insulin and glucagon secretion from pancreatic β- and α-cells, respectively. Dispersed β-cells show cAMP oscillations, but the signaling kinetics in cells within intact islets of Langerhans is unknown. RESEARCH DESIGN AND METHODS The subplasma-membrane cAMP concentration ([cAMP]pm) was recorded in α- and β-cells in the mantle of intact mouse pancreatic islets using total internal reflection microscopy and a fluorescent translocation biosensor. Cell identification was based on the opposite effects of adrenaline on cAMP in α- and β-cells. RESULTS In islets exposed to 3 mmol/L glucose, [cAMP]pm was low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide (GLP-1) induced dose-dependent elevation of [cAMP]pm, often with oscillations synchronized among β-cells. Whereas glucagon also induced [cAMP]pm oscillations in most α-cells, <20% of the α-cells responded to GLP-1. Elevation of the glucose concentration to 11–30 mmol/L in the absence of hormones induced slow [cAMP]pm oscillations in both α- and β-cells. These cAMP oscillations were coordinated with those of the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells but not caused by the changes in [Ca2+]i. The transmembrane adenylyl cyclase (AC) inhibitor 2′5′-dideoxyadenosine suppressed the glucose- and hormone-induced [cAMP]pm elevations, whereas the preferential inhibitors of soluble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-β-triol perturbed cell metabolism and lacked effect, respectively. CONCLUSIONS Oscillatory [cAMP]pm signaling in secretagogue-stimulated β-cells is maintained within intact islets and depends on transmembrane AC activity. The discovery of glucose- and glucagon-induced [cAMP]pm oscillations in α-cells indicates the involvement of cAMP in the regulation of pulsatile glucagon secretion. PMID:21444924

  1. Structural insights into Cn-AMP1, a short disulfide-free multifunctional peptide from green coconut water.

    PubMed

    Santana, Mábio J; de Oliveira, Aline L; Queiroz Júnior, Luiz H K; Mandal, Santi M; Matos, Carolina O; Dias, Renata de O; Franco, Octavio L; Lião, Luciano M

    2015-02-27

    Multifunctional and promiscuous antimicrobial peptides (AMPs) can be used as an efficient strategy to control pathogens. However, little is known about the structural properties of plant promiscuous AMPs without disulfide bonds. CD and NMR were used to elucidate the structure of the promiscuous peptide Cn-AMP1, a disulfide-free peptide isolated from green coconut water. Data here reported shows that peptide structure is transitory and could be different according to the micro-environment. In this regard, Cn-AMP1 showed a random coil in a water environment and an α-helical structure in the presence of SDS-d25 micelles. Moreover, deuterium exchange experiments showed that Gly4, Arg5 and Met9 residues are less accessible to solvent, suggesting that flexibility and cationic charges seem to be essential for Cn-AMP1 multiple activities.

  2. In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease.

    PubMed

    Sprenger, Julia U; Perera, Ruwan K; Steinbrecher, Julia H; Lehnart, Stephan E; Maier, Lars S; Hasenfuss, Gerd; Nikolaev, Viacheslav O

    2015-04-28

    3',5'-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced β-adrenergic receptor-cAMP signalling to SERCA.

  3. Advanced Combustion Modeling for Complex Turbulent Flows

    NASA Technical Reports Server (NTRS)

    Ham, Frank Stanford

    2005-01-01

    The next generation of aircraft engines will need to pass stricter efficiency and emission tests. NASA's Ultra-Efficient Engine Technology (UEET) program has set an ambitious goal of 70% reduction of NO(x) emissions and a 15% increase in fuel efficiency of aircraft engines. We will demonstrate the state-of-the-art combustion tools developed a t Stanford's Center for Turbulence Research (CTR) as part of this program. In the last decade, CTR has spear-headed a multi-physics-based combustion modeling program. Key technologies have been transferred to the aerospace industry and are currently being used for engine simulations. In this demo, we will showcase the next-generation combustion modeling tools that integrate a very high level of detailed physics into advanced flow simulation codes. Combustor flows involve multi-phase physics with liquid fuel jet breakup, evaporation, and eventual combustion. Individual components of the simulation are verified against complex test cases and show excellent agreement with experimental data.

  4. Advanced Microsensors

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This video looks at a spinoff application of the technology from advanced microsensors -- those that monitor and determine conditions of spacecraft like the Space Shuttle. The application featured is concerned with the monitoring of the health of premature babies.

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

    PubMed

    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

    PubMed Central

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

    2013-01-01

    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 Gs α, 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. PMID:22903279

  7. Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis.

    PubMed

    Kumar, Naveen; Gupta, Sonal; Dabral, Surbhi; Singh, Shailja; Sehrawat, Seema

    2017-02-16

    Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP-EPAC1-AKAP9 direction to the development of additional biotherapeutics for breast cancer.

  8. Elevated cyclic AMP levels in T lymphocytes transformed by human T-cell lymphotropic virus type 1.

    PubMed

    Kress, Andrea K; Schneider, Grit; Pichler, Klemens; Kalmer, Martina; Fleckenstein, Bernhard; Grassmann, Ralph

    2010-09-01

    Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4(+) T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1-transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence.

  9. "Store-operated" cAMP signaling contributes to Ca2+-activated Cl- secretion in T84 colonic cells.

    PubMed

    Nichols, Jonathan M; Maiellaro, Isabella; Abi-Jaoude, Joanne; Curci, Silvana; Hofer, Aldebaran M

    2015-10-15

    Apical cAMP-dependent CFTR Cl(-) channels are essential for efficient vectorial movement of ions and fluid into the lumen of the colon. It is well known that Ca(2+)-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca(2+), and the existence of apical Ca(2+)-dependent Cl(-) channels in the native colonic epithelium is controversial, leaving the identity of the Ca(2+)-activated component unresolved. We recently showed that decreasing free Ca(2+) concentration ([Ca(2+)]) within the endoplasmic reticulum (ER) lumen elicits a rise in intracellular cAMP. This process, which we termed "store-operated cAMP signaling" (SOcAMPS), requires the luminal ER Ca(2+) sensor STIM1 and does not depend on changes in cytosolic Ca(2+). Here we assessed the degree to which SOcAMPS participates in Ca(2+)-activated Cl(-) transport as measured by transepithelial short-circuit current (Isc) in polarized T84 monolayers in parallel with imaging of cAMP and PKA activity using fluorescence resonance energy transfer (FRET)-based reporters in single cells. In Ca(2+)-free conditions, the Ca(2+)-releasing agonist carbachol and Ca(2+) ionophore increased Isc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca(2+) chelator BAPTA-AM. The effect on Isc was enhanced in the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), inhibited by the CFTR inhibitor CFTRinh-172 and the PKA inhibitor H-89, and unaffected by Ba(2+) or flufenamic acid. We propose that a discrete component of the "Ca(2+)-dependent" secretory activity in the colon derives from cAMP generated through SOcAMPS. This alternative mode of cAMP production could contribute to the actions of diverse xenobiotic agents that disrupt ER Ca(2+) homeostasis, leading to diarrhea.

  10. AKAP proteins anchor cAMP-dependent protein kinase to KvLQT1/IsK channel complex.

    PubMed

    Potet, F; Scott, J D; Mohammad-Panah, R; Escande, D; Baró, I

    2001-05-01

    In cardiac myocytes, the slow component of the delayed rectifier K(+) current (I(Ks)) is regulated by cAMP. Elevated cAMP increases I(Ks) amplitude, slows its deactivation kinetics, and shifts its activation curve. At the molecular level, I(Ks) channels are composed of KvLQT1/IsK complexes. In a variety of mammalian heterologous expression systems maintained at physiological temperature, we explored cAMP regulation of recombinant KvLQT1/IsK complexes. In these systems, KvLQT1/IsK complexes were totally insensitive to cAMP regulation. cAMP regulation was not restored by coexpression with the dominant negative isoform of KvLQT1 or with the cystic fibrosis transmembrane regulator. In contrast, coexpression of the neuronal A kinase anchoring protein (AKAP)79, a fragment of a cardiac AKAP (mAKAP), or cardiac AKAP15/18 restored cAMP regulation of KvLQT1/IsK complexes inasmuch as cAMP stimulation increased the I(Ks) amplitude, increased its deactivation time constant, and negatively shifted its activation curve. However, in cells expressing an AKAP, the effects of cAMP stimulation on the I(Ks) amplitude remained modest compared with those previously reported in cardiac myocytes. The effects of cAMP stimulation were fully prevented by including the Ht31 peptide (a global disruptor of protein kinase A anchoring) in the intracellular medium. We concluded that cAMP regulation of I(Ks) requires protein kinase A anchoring by AKAPs, which therefore participate with the channel protein complex underlying I(Ks).

  11. Ultrastructural localization of 5'AMP odorant receptor sites on the dendrites of olfactory receptor neurons of the spiny lobster.

    PubMed

    Blaustein, D N; Simmons, R B; Burgess, M F; Derby, C D; Nishikawa, M; Olson, K S

    1993-07-01

    A unique probe--biotinylated adenosine-5'-monophosphate (5'AMP-biotin)--was used in transmission electron microscopic (TEM) studies to localize 5'AMP odorant binding sites on the dendrites of olfactory receptor neurons in the aesthetasc sensilla of the spiny lobster, Panulirus argus. This probe is capable of both binding to and exciting 5'AMP-sensitive olfactory receptor neurons, as revealed through biochemical and electrophysiological assays. TEM studies showed that 5'AMP-biotin binding sites are distributed along the entire dendritic region that is exposed to odorants, including the transitional zone (between the inner and outer dendritic segments, including the ciliary segment) and all of the outer dendritic segment. The density of 5'AMP binding sites per micron2 of membrane is similar along the length of the olfactory dendrite. However, the relative number of 5'AMP-biotin binding sites per micron2 of sensillar area diminishes in the distal 30% of the aesthetasc due to a decrease in the amount of dendritic membrane in that region. The distribution of these 5'AMP binding sites is therefore much more extensive than that of enzymes that inactivate 5'AMP--5'ectonucleotidase/phosphatase--which are restricted to the transitional zone (Gleeson et al., 1991). Taken together, these results suggest that 5'AMP-biotin is labeling 5'AMP-specific olfactory receptor sites that are located along the entire outer dendritic segment and that can be coupled to olfactory transduction. This study represents the first in situ localization of specific olfactory receptor sites using a specific, functionally defined ligand.

  12. “Store-operated” cAMP signaling contributes to Ca2+-