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Sample records for 2-acrylamido-2-methyl-1-propanesulfonic acid amps

  1. Characterization and comparison of methacrylic acid with 2-acrylamido-2-methyl-1-propanesulfonic acid in the preparation of monolithic column for capillary electrochromatography.

    PubMed

    Horiguchi, Daisuke; Ohyama, Kaname; Masunaga, Tomoko; Fujita, Yoshiko; Ali, Marwa Fathy Bakr; Kishikawa, Naoya; Kuroda, Naotaka

    2013-01-01

    Butyl methacrylate (BMA)-ethylene dimethacrylate (EDMA)-methacrylic acid (MAA) and BMA-EDMA-2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monolithic columns were prepared by varying the percentage of ionic monomers for capillary electrochromatography. Monolithic columns with a higher content of ionic monomers provided better column efficiency, and the performance of BMA-EDMA-MAA monoliths was better than BMA-EDMA-AMPS. To characterize and optimize BMA-EDMA-MAA monoliths, the effects of the content of cross-linker and the total monomer in the polymerization mixture on column performance were also studied. Plate heights of 8.2 µm for the unretained solute (thiourea) and 12.6 µm for the retained solute (naphthalene) were achieved with a monolithic column using 2.5% MAA (Column I).

  2. SYNTHESIS AND IN VITRO CHARACTERIZATION OF HYDROXYPROPYL METHYLCELLULOSE-GRAFT-POLY (ACRYLIC ACID/2-ACRYLAMIDO-2-METHYL-1-PROPANESULFONIC ACID) POLYMERIC NETWORK FOR CONTROLLED RELEASE OF CAPTOPRIL.

    PubMed

    Furqan Muhammad, Iqbal; Mahmood, Ahmad; Aysha, Rashid

    2016-01-01

    A super-absorbent hydrogel was developed by crosslinking of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and acrylic acid with hydroxypropyl methylcellulose (HPMC) for controlled release drug delivery of captopril, a well known antihypertensive drug. Acrylic acid and AMPS were polymerized and crosslinked with HPMC by free radical polymerization, a widely used chemical crosslinking method. N,N'-methylenebisacrylamide (MBA) and potassium persulfate (KPS) were added as cross-linker and initiator, respectively. The hydrogel formulation was loaded with captopril (as model drug). The concentration of captopril was monitored at 205 nm using UV spectrophotometer. Equilibrium swelling ratio was determined at pH 2, 4.5 and 7.4 to evaluate the pH responsiveness of the formed hydrogel. The super-absorbent hydrogels were evaluated by FTIR, SEM, XRD, and thermal analysis (DSC and TGA). The formation of new copolymeric network was determined by FTIR, XRD, TGA and DSC analysis. The hydrogel formulations with acrylic acid and AMPS ratio of 4: 1 and lower amounts of crosslinker had shown maximum swelling. Moreover, higher release rate of captopril was observed at pH 7.4 than at pH 2, because of more swelling capacity of copolymer with increasing pH of the aqueous medium. The present research work confirms the development of a stable hydrogel comprising of HPMC with acrylic acid and AMPS. The prepared hydrogels exhibited pH sensitive behav-ior. This superabsorbent composite prepared could be a successful drug carrier for treating hypertension.

  3. 2-Acrylamido-2-methyl-1-propanesulfonic Acid Grafted Poly(vinylidene fluoride-co-hexafluoropropylene)-Based Acid-/Oxidative-Resistant Cation Exchange for Membrane Electrolysis.

    PubMed

    Pandey, Ravi P; Das, Arindam K; Shahi, Vinod K

    2015-12-30

    For developing acid-/oxidative-resistant aliphatic-polymer-based cation-exchange membrane (CEM), macromolecular modification of poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-co-HFP) was carried out by controlled chemical grafting of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS). To introduce the unsaturation suitable for chemical grafting, dehydrofluorination of commercially available PVDF-co-HFP was achieved under alkaline medium. Sulfonated copolymer (SCP) was prepared by the free radical copolymerization of dehydofluorinated PVDF-co-HFP (DHPVDF-co-HFP) and AMPS in the presence of free radical initiator. Prepared SCP-based CEMs were analyzed for their morphological characteristics, ion-exchange capacity (IEC), water uptake, conductivity, and stabilities (mechanical, chemical, and thermal) in comparison with state-of-art Nafion117 membrane. High bound water content avoids the membrane dehydration, and most optimal (SCP-1.33) membrane exhibited about ∼2.5-fold high bound water content in comparison with that of Nafion117 membrane. Bunsen reaction of iodine-sulfur (I-S) was successfully performed by direct-contact-mode membrane electrolysis in a two-compartment electrolytic cell using different SCP membranes. High current efficiency (83-99%) confirmed absence of any side reaction and 328.05 kJ mol-H2(-1) energy was required for to produce 1 mol of H2 by electrolytic cell with SCP-1.33 membrane. In spite of low conductivity for reported SCP membrane in comparison with that of Nafion117 membrane, SCP-1.33 membrane was assessed as suitable candidate for electrolysis because of its low-cost nature and excellent stabilities in highly acidic environment may be due to partial fluorinated segments in the membrane structure.

  4. A novel organic/inorganic polymer membrane based on poly(vinyl alcohol)/poly(2-acrylamido-2-methyl-1-propanesulfonic acid/3-glycidyloxypropyl trimethoxysilane polymer electrolyte membrane for direct methanol fuel cells

    NASA Astrophysics Data System (ADS)

    Yang, Chun-Chen; Lue, Shingjiang Jessie; Shih, Jeng-Ywan

    2011-05-01

    Poly(vinyl alcohol)/poly(2-acrylamido-2-methyl-1-propanesulfonic acid (PAMPS)/3-glycidyloxypropyl)trimethoxysilane (PVA/PAMPS/GPTMS) organic/inorganic proton-conducting polymer membranes are prepared by a solution casting method. PAMPS is a polymeric acid commonly used as a primary proton donor, while 3-(glycidyloxypropyl)trimethoxysilane (GPTMS) is an inorganic precursor forming a semi-interpenetrating network (SIPN). Varying amounts of sulfosuccinic acid (SSA) are used as the cross-linker and secondary proton source. The characteristic properties of PVA/PAMPS/GPTMS composite membranes are investigated by thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), micro-Raman spectroscopy and the AC impedance method. Direct methanol fuel cells (DMFCs) made of PVA/PAMPS/GPTMS composite membranes are assembled and examined. Experimental results indicate that DMFCs employing an inexpensive, non-perfluorinated, organic/inorganic SIPN membrane achieve good electrochemical performance. The highest peak power density of a DMFC using PVA/PAMPS/GPTMS composite membrane with 2 M CH3OH solution fuel at ambient temperature is 23.63 mW cm-2. The proposed organic/inorganic proton-conducting membrane based on PVA/PAMPS/GPTMS appears to be a viable candidate for future DMFC applications.

  5. New core@shell nanogel based 2-acrylamido-2-methyl-1-propane sulfonic acid for preconcentration of Pb(II) from various water samples

    NASA Astrophysics Data System (ADS)

    Shoueir, Kamel Rizq; Akl, Magda Ali; Sarhan, Ali Ali; Atta, Ayman Mohamdy

    2016-12-01

    Poly(vinyl alcohol) core coated with poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-N-isopropylacrylamide) shell to produce well-define PVA@P(AMPS-co-NIPAm) core shell nanogels with a core of 25 ± 0.5 nm and shell of 5 ± 0.5 nm. The synthetic approach was produced by a surfactant free emulsion polymerization (SFEP). The specific area was found to be 1685.8 m2/g. The nanogels were studied in a batch adsorption for removal of Pb(II) ions and characterized by SEM, TEM, TGA and BET measurements. The results showed that the adsorption equilibrium data fitted the Langmuir isotherm and the kinetic studies are well described by the pseudo-second-order kinetic model. The Pb(II) maximum adsorption was 510.2 (mg/g) for PVA@P(90AMPS-co-10NIPAm) (wt.: wt%). The PVA@P(AMPS-co-NIPAm) nanogels were applied for extracting of Pb(II) in real different environmental water samples successfully with high recoveries reaches 104.4%.

  6. Poly(NIPAm-AMPS) nanoparticles for targeted delivery of anti-inflammatory cell penetrating peptides

    NASA Astrophysics Data System (ADS)

    Bartlett, Rush Lloyd, II

    Inflammatory diseases such as osteoarthritis and rheumatoid arthritis cause $127.8 billion in US healthcare expenditures each year and are the cause of disability for 27% of disabled persons in the United States. Current treatment options rarely halt disease progression and often result in significant unwanted and debilitating side effects. Our laboratory has previously developed a family of cell penetrating peptides (CPPs) which inhibit the activity of mitogen activated protein kinase activate protein kinase 2 (MK2). MK2 mediates the inflammatory response by activating Tristetraprline (TTP). Once activated, TTP rapidly stabilizes AU rich regions of pro-inflammatory cytokine mRNA which allows translation of pro-inflammatory cytokines to occur. Blocking MK2 with our labs CPPs yields a decrease in inflammatory activity but CPPs by are highly non specific and prone to rapid enzymatic degradation in vivo.. In order to increase the potency of MK2 inhibiting CPPs we have developed a novel nanoparticle drug carrier composed of poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid). This drug carrier has been shown to have preliminary efficacy in vitro and ex vivo for suppressing pro-inflammatory cytokine production when releasing CPPs. This thesis will present progress made on three aims: Specific Aim 1) Create and validate a NIPAm based drug delivery system that mimics the binding and release previously observed between cell penetrating peptides and glycosaminoglycans. Specific Aim 2) Engineer degradability into poly(NIPAm-AMPS) nanoparticles to enable more drug to be released and qualify that system in vitro. Specific Aim 3) Validate poly(NIPAm-AMPS) nanoparticles for targeted drug delivery in an ex vivo inflammatory model. Overall we have developed a novel anionic nanoparticle system that is biocompatible and efficient at loading and releasing cell penetrating peptides to inflamed tissue. Once loaded with a CPP the nanoparticle drug complex is

  7. Charge storage in polymer acid-doped polyaniline-based layer-by-layer electrodes.

    PubMed

    Jeon, Ju-Won; O'Neal, Josh; Shao, Lin; Lutkenhaus, Jodie L

    2013-10-23

    Polymeric electrodes that can achieve high doping levels and store charge reversibly are desired for electrochemical energy storage because they can potentially achieve high specific capacities and energies. One such candidate is the polyaniline:poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PANI:PAAMPSA) complex, a water-processable complex obtained via template polymerization that is known to reversibly achieve high doping levels at potentials of up to 4.5 V versus Li/Li+. Here, for the first time, PANI:PAAMPSA is successfully incorporated into layer-by-layer (LbL) electrodes. This processing technique is chosen for its ability to blend species on a molecular level and its ability to conformally coat a substrate. Three different polyaniline-based LbL electrodes comprised of PANI/PAAMPSA, PANI/PANI:PAAMPSA, and linear poly(ethylenimine)/PANI:PAAMPSA are compared in terms of film growth, charge storage, and reversibility. We found that the reversibility of PANI:PAAMPSA is retained within the LbL electrodes and that the PANI/PANI:PAAMPSA electrode exhibits the best performance in terms of capacity and cycle life. These results provide general guidelines for the assembly of PANI:PAAMPSA in LbL films and also demonstrate their potential as electrochemically active components in electrodes.

  8. [Preparation of monolithic polylaurylmethacrylate column and its application in capillary electrochromatographic separation of myoglobin digests].

    PubMed

    Wang, Tingting; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2010-03-01

    The monolithic polylaurylmethacrylate column was prepared in a single step using the monomer solution containing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA), and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), with the ternary porogenic solvent consisting of 1-propanol, 1,4-butanediol and H2O. The effects of AMPS content and the ratio of monomer solution to porogenic solvent were investigated. The optimal mass ratio of monomer solution to porogenic solvent was 35:65, the monomer solution was composed of 59.5% (w/w) LMA, 40% (w/w) EDMA and 0.5% (w/w) AMPS, and the porogenic solution was composed of 60% (w/w) 1-propanol, 30% (w/w) 1,4-butanediol and 10% (w/w) H2O. The prepared monolithic column was successfully applied in the capillary electrochromatographic (CEC) separation of myoglobin digests under the optimized mobile phase.

  9. High yield sample preconcentration using a highly ion-conductive charge-selective polymer.

    PubMed

    Chun, Honggu; Chung, Taek Dong; Ramsey, J Michael

    2010-07-15

    The development and analysis of a microfluidic sample preconcentration system using a highly ion-conductive charge-selective polymer [poly-AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid)] is reported. The preconcentration is based on the phenomenon of concentration polarization which develops at the boundaries of the poly-AMPS with buffer solutions. A negatively charged polymer, poly-AMPS, positioned between two microchannels efficiently extracts cations through its large cross section, resulting in efficient anion sample preconcentration. The present work includes the development of a robust polymer that is stable over a wide range of buffers with varying chemical compositions. The sample preconcentration effect remains linear to over 3 mM (0.15 pmol) and 500 microM (15 fmol) for fluorescein and TRITC-tagged albumin solutions, respectively. The system can potentially be used for concentrating proteins on microfluidic devices with subsequent analysis for proteomic applications.

  10. Superabsorbent nanocomposite (alginate-g-PAMPS/MMT): synthesis, characterization and swelling behavior.

    PubMed

    Yadav, Mithilesh; Rhee, Kyong Yop

    2012-09-01

    A superabsorbent composite (alginate-g-PAMPS/MMT) was prepared by graft copolymerization from alginate, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and Na+ montmorillonite (MMT) in an inert atmosphere. Effects of polymerization variables on water absorbency, including the content of Na+ montmorillonite, sodium alginate, N,N'-methylenebisacrylamide and AMPS, were studied. The introduced montmorillonite formed a loose and porous surface and improved the water absorbency of the alginate-g-PAMPS/MMT superabsorbent composite. Swelling behaviors of the superabsorbent composites in various cationic salt solutions (NaCl, CaCl2 and FeCl3) and anionic salt solutions (NaCl and Na2SO4) were also systematically investigated. The superabsorbent composite was further characterized using Fourier transform infrared spectroscopy (FTIR), rheology, thermogravimetric analysis (TGA), X-ray diffraction (XRD) and scanning electron microscopy (SEM) taking alginate-g-PAMPS as a reference.

  11. Preparation and characterization of polymer electrolyte membranes based on silicon-containing core-shell structured nanocomposite latex particles

    NASA Astrophysics Data System (ADS)

    Zhong, Shuangling; Sun, Chenggang; Gao, Yushan; Cui, Xuejun

    2015-09-01

    A series of silicon-containing core-shell structured polyacrylate/2-acrylamido-2-methyl-1-propanesulfonic acid (SiO2-CS-PA/A) nanocomposite latex particles are prepared by the emulsifier-free emulsion polymerization of acrylate monomers and various amount of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) with colloidal nanosilica particles as seed. The chemical and morphological structures of latex particles with high monomer conversion are determined using Fourier transform infrared (FTIR), dynamic light scattering (DLS) and transmission electron microscopy (TEM). The SiO2-CS-PA/A nanocomposite membranes are fabricated through pouring the latex onto a clean surface of glass and drying at 60 °C for 10 h and 120 °C for 2 h. The nanocomposite membranes possess good thermal and dimensional stability. In addition, in comparison to Nafion® 117, the nanocomposite membranes exhibit moderate proton conductivity, significantly better methanol barrier and selectivity. The methanol diffusion coefficient is in the range of 1.03 × 10-8 to 5.26 × 10-8 cm2 s-1 which is about two orders of magnitude lower than that of Nafion® 117 (2.36 × 10-6 cm2 s-1). The SiO2-CS-PA/A 5 membrane shows the highest selectivity value (2.34 × 105 S cm-3) which is approximately 11.0 times of that (2.13 × 104 S cm-3) of Nafion® 117. These results indicate that the nanocomposite membranes are promising candidates to be used as polymer electrolyte membranes in direct methanol fuel cells.

  12. A doped polyaniline modified electrode amperometric biosensor for gluconic acid determination in grapes.

    PubMed

    Albanese, Donatella; Malvano, Francesca; Sannini, Adriana; Pilloton, Roberto; Di Matteo, Marisa

    2014-06-23

    In winemaking gluconic acid is an important marker for quantitative evaluation of grape infection by Botrytis cinerea. A screen-printed amperometric bienzymatic sensor for the determination of gluconic acid based on gluconate kinase (GK) and 6-phospho-D-gluconate dehydrogenase (6PGDH) coimmobilized onto polyaniline/poly (2-acrylamido-2-methyl-1-propanesulfonic acid; PANI-PAAMPSA) is reported in this study. The conductive polymer electrodeposed on the working electrode surface allowed the detection of NADH at low potential (0.1 V) with a linear range from 4 × 10(-3) to 1 mM (R2 = 0.99) and a sensitivity of 419.44 nA∙mM(-1). The bienzymatic sensor has been optimized with regard to GK/6PGDH enzymatic unit ratio and ATP/NADP+ molar ratio which resulted equal to 0.33 and 1.2, respectively. Under these conditions a sensitivity of 255.2 nA∙mM(-1), a limit of detection of 5 μM and a Relative Standard Deviation (RSD) of 4.2% (n = 5) have been observed. Finally, the biosensor has been applied for gluconic acid measurements in must grape samples and the matrix effect has been taken into consideration. The results have been compared with those obtained on the same samples with a commercial kit based on a spectrophotometric enzyme assay and were in good agreement, showing the capability of the bienzymatic PANI-PAAMPSA biosensor for gluconic acid measurements and thus for the evaluation of Botrytis cinerea infection in grapes.

  13. Sulfonated acrylamide copolymers as pseudo-stationary phases in electrokinetic chromatography.

    PubMed

    Shi, W; Watson, C J; Palmer, C P

    2001-01-05

    Sulfonated copolymers were synthesized, characterized and used as separation media in electrokinetic chromatography. The polymers used were synthesized from AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid) and LMAm (lauryl methacrylamide) in different mole ratios (from 100:0 to 60:40). Electrophoretic mobilities and methylene selectivities were calculated, which showed the expected correlation with the monomer ratios. The chemical selectivities for the separation of nine solutes by the copolymers were compared with that of sodium lauryl sulfate micelles, showing significant differences. No significant difference in chemical selectivities was observed for copolymers with different monomer ratios. No significant change of hydrophobic microdomain of copolymers was found in background buffers with different ionic strength values, based on the investigation of the retention factors, methylene selectivities and polymer effective mobilities. No change of hydrophobic microdomain of the copolymer solutions was found at copolymer concentrations from 0.17 to 3% (w/v), however, plots of k' versus polymer concentration suggested a different copolymer phase at lower concentrations (from 0 to 0.1%, w/v) from that at higher concentrations (from 0.17 to 3%, w/v). The copolymer with AMPS-LMAm (80:20) could be chosen as optimum copolymer as far as the methylene selectivity, peak symmetry and polymer mobility were concerned.

  14. Comparison of positively and negatively charged achiral co-monomers added to cyclodextrin monolith: improved chiral separations in capillary electrochromatography.

    PubMed

    Lu, Yang; Shamsi, Shahab A

    2014-10-01

    Cyclodextrins (CDs) and their derivatives have been one of the most popular and successful chiral additives used in electrokinetic chromatography because of the presence of multiple chiral centers, which leads to multiple chiral interactions. However, there has been relatively less published work on the use of CDs as monolithic media for capillary electrochromatography (CEC). The goal of this study was to show how the addition of achiral co-monomer to a polymerizable CD such as glycidyl methacrylate β-cyclodextrin (GMA/β-CD) can affect the enantioselective separations in monolithic CEC. To achieve this goal, polymeric monoliths columns were prepared by co-polymerizing GMA/β-CD with cationic or anionic achiral co-monomers [(2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and vinyl benzyltrimethyl-ammonium (VBTA)] in the presence of conventional crosslinker (ethylene dimethacrylate) and ternary porogen system including butanediol, propanol and water. A total of 34 negatively charged compounds, 30 positively charged compounds and 33 neutral compounds were screened to compare the enantioresolution capability on the GMA/β-CD, GMA/β-CD-VBTA and GMA/β-CD-AMPS monolithic columns.

  15. Comparison of Positively and Negatively Charged Achiral Co-Monomers Added to Cyclodextrin Monolith: Improved Chiral Separations in Capillary Electrochromatography

    PubMed Central

    Lu, Yang; Shamsi, Shahab A.

    2014-01-01

    Cyclodextrins (CDs) and their derivatives have been one of the most popular and successful chiral additives used in electrokinetic chromatography because of the presence of multiple chiral centers, which leads to multiple chiral interactions. However, there has been relatively less published work on the use of CDs as monolithic media for capillary electrochromatography (CEC). The goal of this study was to show how the addition of achiral co-monomer to a polymerizable CD such as glycidyl methacrylate β-cyclodextrin (GMA/β-CD) can affect the enantioselective separations in monolithic CEC. To achieve this goal, polymeric monoliths columns were prepared by co-polymerizing GMA/β-CD with cationic or anionic achiral co-monomers [(2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and vinyl benzyltrimethyl-ammonium (VBTA)] in the presence of conventional crosslinker (ethylene dimethacrylate) and ternary porogen system including butanediol, propanol and water. A total of 34 negatively charged compounds, 30 positively charged compounds and 33 neutral compounds were screened to compare the enantioresolution capability on the GMA/β-CD, GMA/β-CD-VBTA and GMA/β-CD-AMPS monolithic columns. PMID:24108813

  16. Immobilization of Stimuli-Responsive Nanogels onto Honeycomb Porous Surfaces and Controlled Release of Proteins.

    PubMed

    De León, A S; Molina, M; Wedepohl, S; Muñoz-Bonilla, A; Rodríguez-Hernández, J; Calderón, M

    2016-02-23

    In this article, we describe the formation of functional honeycomb-like porous surfaces fabricated by the breath figures technique using blends of either amino-terminated poly(styrene) or a poly(styrene)-b-poly(acrylic acid) block copolymer with homopoly(styrene). Thus, the porous interfaces exhibited either amino or acid groups selectively located inside of the holes, which were subsequently employed to anchor stimuli-responsive nanogels by electrostatic interactions. These nanogels were prepared from poly(N-isopropylacrylamide) (PNIPAM) cross-linked with dendritic polyglycerol (dPG) and semi-interpenetrated with either 2-(dimethylamino)ethyl methacrylate (DMAEMA) or 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) to produce positively and negatively charged nanogel surfaces, respectively. The immobilization of these semi-interpenetrated networks onto the surfaces allowed us to have unique stimuli-responsive surfaces with both controlled topography and composition. More interestingly, the surfaces exhibited stimuli-responsive behavior by variations on the pH or temperature. Finally, the surfaces were evaluated regarding their capacity to induce a thermally triggered protein release at temperatures above the cloud point temperature (T(cp)) of the nanogels.

  17. Adhesion, Proliferation and Migration of NIH/3T3 Cells on Modified Polyaniline Surfaces

    PubMed Central

    Rejmontová, Petra; Capáková, Zdenka; Mikušová, Nikola; Maráková, Nela; Kašpárková, Věra; Lehocký, Marián; Humpolíček, Petr

    2016-01-01

    Polyaniline shows great potential and promises wide application in the biomedical field thanks to its intrinsic conductivity and material properties, which closely resemble natural tissues. Surface properties are crucial, as these predetermine any interaction with biological fluids, proteins and cells. An advantage of polyaniline is the simple modification of its surface, e.g., by using various dopant acids. An investigation was made into the adhesion, proliferation and migration of mouse embryonic fibroblasts on pristine polyaniline films and films doped with sulfamic and phosphotungstic acids. In addition, polyaniline films supplemented with poly (2-acrylamido-2-methyl-1-propanesulfonic) acid at various ratios were tested. Results showed that the NIH/3T3 cell line was able to adhere, proliferate and migrate on the pristine polyaniline films as well as those films doped with sulfamic and phosphotungstic acids; thus, utilization of said forms in biomedicine appears promising. Nevertheless, incorporating poly (2-acrylamido-2-methyl-1-propanesulfonic) acid altered the surface properties of the polyaniline films and significantly affected cell behavior. In order to reveal the crucial factor influencing the surface/cell interaction, cell behavior is discussed in the context of the surface energy of individual samples. It was clearly demonstrated that the lesser the difference between the surface energy of the sample and cell, the more cyto-compatible the surface is. PMID:27649159

  18. Using scanning contactless conductivity to optimise photografting procedures and capacity in the production of polymer ion-exchange monoliths.

    PubMed

    Gillespie, Eoin; Connolly, Damian; Paull, Brett

    2009-07-01

    Capacitively coupled contactless conductivity detection (C4D) is utilised as a simple, rapid and non-invasive technique for the quantitative evaluation of the ion-exchange capacity of charged polymer monoliths in capillary format. A charged monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) was photografted onto a 100 microm i.d. butyl methacrylate-co-ethylenedimethacrylate monolith in a number of discrete 10 mm zones. By varying the energy dose (J/cm2) during grafting of each zone, the grafting density and thus ion-exchange capacity could be precisely controlled. Ion-exchange capacity could be correlated with energy dose by measuring the conductive response of each grafted region using scanning C4D techniques. Repeatability of the scanning C4D method was excellent with % RSD values of 0.7% and 2.4% obtained for three replicate scans of the ungrafted and grafted regions of a single monolith, respectively. Repeatability of the photografting process on separate monoliths was also examined by comparison of C4D profiles. The spatial accuracy of photografting was probed using scanning C4D which could measure the conductive response of the monolith at measurement intervals as low as 1 mm along its entire length. Scanning C4D was also used for the real time visualisation of the equilibration of grafted zones to permit the optimisation of monolith washing procedures. Finally, scanning C4D was applied to the measurement of the ion-exchange capacity of butyl methacrylate-co-AMPS-co-ethylenedimethacrylate copolymers with a direct correlation between monolith conductive response and concentration of charged monomer in the polymerisation mixture. The longitudinal homogeneity of charge along the monolith was 0.3% RSD, demonstrating that the charged functional monomer was evenly dispersed throughout the bulk of the monolith. Ion-exchange capacity was cross validated chromatographically using breakthrough studies and found to closely correlate to within 1% of the

  19. Targeted Smart pH and Thermoresponsive N,O-Carboxymethyl Chitosan Conjugated Nanogels for Enhanced Therapeutic Efficacy of Doxorubicin in MCF-7 Breast Cancer Cells.

    PubMed

    Verma, Neeraj K; Purohit, Mahaveer P; Equbal, Danish; Dhiman, Nitesh; Singh, Amrita; Kar, Aditya K; Shankar, Jai; Tehlan, Sarita; Patnaik, Satyakam

    2016-11-16

    In cancer treatment, developing ideal anticancer drug delivery systems to target tumor microenvironment by circumventing various physiological barriers still remains a daunting challenge. Here, in our work, a series of pH- and temperature-responsive nanogels based on poly(N-isopropylacrylamide-co-1-propene-2-3-dicarboxylate-co-2-acrylamido-2-methyl-1-propanesulfonate [poly(NIPAAm-IA-AMPS)] cross-linked by ethylene glycol dimethacrylate (EGDMA) were synthesized by random copolymerization. The molar ratio between monomer-comonomers-cross-linker was varied to fine-tune the optimum responsiveness of the nanogels. These optimized nanogels were further coupled to N,O-carboxymethyl chitosan (NOCC) stoichiometrically using EDC-NHS coupling chemistry to enhance the swelling behavior at lower pH. Interestingly, these NOCC-g-nanogels, when dispersed in aqueous media under sonication, attain nanosize and retain their high water-retention capacity with conspicuous pH and temperature responsiveness (viz. nanogel shrinkage in size beyond 35 °C and swelled at acidic pH) in vitro, as reflected by dynamic light scattering data. Doxorubicin (DOX), a potent anticancer drug, was loaded into these nanogels using the physical entrapment method. These drug-loaded nanogels exhibited a slow and sustained DOX release profile at physiological temperature and cytosolic pH. Furthermore, confocal and TEM results demonstrate that these nanogels were swiftly internalized by MCF-7 cells, and cell viability data showed preferential heightened cytotoxicity toward cancer cells (MCF-7 and MDA-MB231) compared to the MCF10A cells (human breast epithelial cell). Furthermore, intracellular DNA damage and cell cycle arrest assays suggest a mitochondrial mediated apoptosis in MCF-7 cells. This study substantiates our NOCC-g-nanogel platform as an excellent modality for passive diffusive loading and targeted release of entrapped drug(s) at physiological conditions in a controlled way for the improved

  20. Superabsorbent, High Porosity, PAMPS-Based Hydrogels through Emulsion Templating.

    PubMed

    Kovačič, Sebastijan; Silverstein, Michael S

    2016-09-26

    Swell! Superabsorbent, mechanically robust, high-porosity hydrogels based on poly(2-acrylamido-2-methyl-1-propanesulfonic acid) have been successfully synthesized by templating within high internal phase emulsions (HIPEs). These hydrogel polyHIPEs (HG-PHs) exhibit unusually high uptakes of water and of artificial urine through structure- and crosslinking-dependent hydrogel-swelling-driven void expansion. An HG-PH with 3.1 mmol g(-1) of highly accessible sulfonic acid groups exhibits a 7 meq NaOH ion exchange capacity per gram polymer and rapid dye absorption. The highly swollen HG-PHs do not fail at compressive strains of up to 60%, they retain water and recover their shapes upon the removal of stress. Unusually, the dry hydrogels have relatively high compressive moduli and achieve relatively high stresses at 70% strain.

  1. Removal of bovine serum albumin using solid-phase extraction with in-situ polymerized stationary phase in a microfluidic device.

    PubMed

    Lee, Eun Zoo; Huh, Yun Suk; Jun, Young-Si; Won, Hyo Jin; Hong, Yeon Ki; Park, Tae Jung; Lee, Sang Yup; Hong, Won Hi

    2008-04-11

    Serum albumin, one of the most abundant serum proteins, blocks the expression of other important biomarkers. The objective of this study is to remove serum albumin effectively by using solid-phase extraction (SPE) in microfluidic devices. Photo-polymerized adsorbent as a stationary phase of SPE was used to remove bovine serum albumin (BSA). The adsorption capacity was examined with the effect of pH and concentration in BSA solution, and adjustment of monomer concentration such as hydrophilic 2-acrylamido-2-methyl-1-propanesulfonic acid and acrylamide in the adsorbent. The effect of hydrophobic butyl methacylate on BSA adsorption was also studied. Selective removal in a bicomponent with BSA and bovine gamma-globulin was performed by adjusting the pH as required.

  2. Influence of polyelectrolyte on the thermosensitive property of PNIPAAm-based copolymer hydrogels.

    PubMed

    Zhang, Xian-Zheng; Chu, Chih-Chang

    2007-09-01

    A new family of poly(NIPAAm-co-2-acrylamido-2-methyl-1-propanesulfonic acid) [P(NIPAAm-co-AMPSA)] hydrogels was synthesized by incorporating negative charged AMPSA to the backbone of the PNIPAAm-based hydrogel. The effect of polyelectrolyte (i.e., PAMPSA) on the thermosensitive property of PNIPAAm hydrogels was investigated. It was found that P(NIPAAm-co-AMPSA) hydrogels exhibited unique honey-comb-like 3D porous structure having rigid cell wall as well as enhanced mechanical property. The incorporation of AMPSA into PNIPAAm backbones also led to a significant increase in swelling capability at room temperature when comparing to pure PNIPAAm hydrogels. In addition, the shrinking rate upon heating was significantly improved if the AMPSA content in P(NIPAAm-co-AMPSA) hydrogels was less than 10 wt%.

  3. "Smart" polymeric nanospheres as new materials for possible biomedical applications.

    PubMed

    Szczubiałka, Krzysztof; Jankowska, Monika; Nowakowska, Maria

    2003-08-01

    Novel random terpolymers of N-isopropylacrylamide (NIPAM), sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), and cinnamoyloxyethylmethacrylate (CEMA) were synthesized by free radical copolymerization using AIBN as an initiator. Five terpolymers were obtained by copolymerization of the monomer mixtures containing a fixed amount of 10 mol % of AMPS while the content of CEMA ranged from 5 to 25 mol % and was changed in 5 mol % increments. The terpolymers obtained are water-soluble. Because of their amphiphilic nature they undergo self-organization in the aqueous solution with the formation of micelles capable of solubilizing sparingly water soluble organic compounds, such as drugs. The terpolymers are susceptible to three external stimuli, i.e. temperature, ionic strength and UV light. Due to the presence of NIPAM in the terpolymers they display the lower critical solution temperature (LCST), the presence of AMPS makes them sensitive to the ionic strength of the solution, while the light-responsiveness of the terpolymers is due to the presence of cinnamoyl chromophores, which undergo photodimerization when irradiated with UV light at about 280 nm. Application of any of these stimuli alone or in combination with other stimuli allows changing the copolymer properties in a controlled way.

  4. Polymer monoliths with low hydrophobicity for strong cation-exchange capillary liquid chromatography of peptides and proteins.

    PubMed

    Gu, Binghe; Li, Yun; Lee, Milton L

    2007-08-01

    Two polymer monoliths were designed and synthesized from commercially available monomers with an attempt to decrease hydrophobicity for strong cation-exchange chromatography. One was prepared from the copolymerization of sulfoethyl methacrylate and poly(ethylene glycol) diacrylate, and the other was synthesized from vinylsulfonic acid and poly(ethylene glycol) diacrylate. Both of the monoliths were synthesized inside 75-microm i.d., UV-transparent fused-silica capillaries by photopolymerization. The hydrophobicities of the two monoliths were systematically evaluated using standard synthetic undecapeptides under ion-exchange conditions and propyl paraben under reversed-phase conditions. The poly(sulfoethyl methacrylate) monolith demonstrated similar hydrophobicity as a monolith prepared from copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid and poly(ethylene glycol) diacrylate, and 40% acetonitrile was required to suppress any hydrophobic interactions with peptides under ion-exchange conditions. However, with the use of vinylsulfonic acid as the functional monomer, a monolith with very low hydrophobicity was obtained, making it suitable for strong cation-exchange liquid chromatography of both peptides and proteins. It was found that monolith hydrophobicity could be adjusted by selection of monomers that differ in hydrocarbon content and type of vinyl group. Finally, excellent separations of model protein standards and high-density lipoproteins were achieved using the poly(vinylsulfonic acid) monolith. Five subclasses of high-density lipoproteins were resolved using a simple linear NaCl gradient.

  5. Exploiting the Different Polarity in Piezoresistive Characteristics of Conducting Polymers for Strain Gauge Applications

    NASA Astrophysics Data System (ADS)

    Sezen, Melda; Register, Jeffrey T.; Yao, Yao; Glisic, Branko; Loo, Yueh-Lin

    2015-03-01

    Piezoresistivity defines the change in resistance of a material in response to mechanical stress. We exploited the effects of structural modifications on the piezoresistive properties of conducting polymers, poly(2-acrylamido-2-methyl-1-propanesulfonic acid) doped polyaniline, PANI-PAAMPSA, and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS, for strain gauge applications. Under tensile deformation, the resistances of as-cast PANI-PAAMPSA and PEDOT:PSS increase due to increased separation between the electrostatically stabilized conducting polymer particles. Upon solvent annealing in dichloroacetic acid, DCA, PANI-PAAMPSA's resistance decreases whereas PEDOT:PSS's resistance still increases with tension. While DCA treatment reduces the electrostatic interactions between PANI and PAAMPSA, it only removes the PSS overlayer in PEDOT:PSS. The change in the polarity of PANI-PAAMPSA's piezoresistivity is attributed to the unlocking of the globular structure of the as-synthesized conducting polymer complex with DCA-treatment, which then enables strain-induced crystallization on deformation. By tuning the piezoresistive characteristics of the polymers through structural modification, we can design strain gauge circuits for monitoring the conditions of civil structures.

  6. Synthesis of magnetic composite nanoparticles enveloped in copolymers specified for scale inhibition application

    NASA Astrophysics Data System (ADS)

    Do, Bao Phuong Huu; Dung Nguyen, Ba; Duy Nguyen, Hoang; Nguyen, Phuong Tung

    2013-12-01

    We report the synthesis of magnetic iron oxide nanoparticles encapsulated in maleic acid-2-acrylamido-2-methyl-1-propanesulfonate based polymer. This composite nanoparticle is specified for the high-pressure/high-temperature (HPHT) oilfield scale inhibition application. The process includes a facile-ultrasound-supported addition reaction to obtain iron oxide nanoparticles with surface coated by oleic acid. Then via inverse microemulsion polymerization with selected monomers, the specifically designed copolymers have been formatted in nanoscale. The structure and morphology of obtained materials were characterized by transmission electron microscopy (TEM), x-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and the thermal stability. The effectiveness of synthesized compounds as a carbonate scale inhibitor was investigated by testing method NACE standard TM 03-074-95 at aging temperature of 70, 90 and 120 °C. The magnetic nanocomposite particles can be easily collected and detected demonstrating their superior monitoring ability, which is absent in the case of conventional copolymer-based scale inhibitor.

  7. CEC with new monolithic stationary phase based on a fluorinated monomer, trifluoroethyl methacrylate.

    PubMed

    Yurtsever, Arda; Saraçoğlu, Berna; Tuncel, Ali

    2009-02-01

    A new, fluorinated monolithic stationary phase for CEC was first synthesized by a single-stage, thermally initiated copolymerization of a fluorinated monomer, 2,2,2-trifluoroethyl methacrylate (TFEM) and ethylene dimethacrylate (EDMA) in the presence of a porogen mixture. In this preparation, 2-acrylamido-2-methyl-1-propanesulfonic acid was used as the charge-bearing monomer. The porogen mixture was prepared by mixing isoamylalcohol and 1,4-butanediol. A clear increase in the electroosmotic mobility was observed with increasing pH. The electroosmotic mobility decreased with increasing ACN concentration. Poly(TFEM-co-EDMA) monolith prepared under optimized polymerization conditions was successfully used in the separation of alkylbenzenes and phenols by CEC. The best chromatographic separation for alkylbenzenes was performed with lower ACN concentrations (i.e. 60% v/v) with respect to the common acrylic-based CEC monoliths. The theoretical plate numbers up to 220 000 plates/m were achieved in the reversed phase separation of phenols. Poly(TFEM-co-EDMA) monolith also allowed the simultaneous separation of aniline and benzoic acid derivatives by a single run and by using a lower ACN concentration in the mobile phase with respect to the similar electrochromatographic separations. A stable retention behaviour in reversed phase separation of alkylbenzenes was obtained with the poly(TFEM-co-EDMA) monolith.

  8. Facile fabrication of hierarchical ZnO microstructures assisted with PAMPSA and enhancement of green emission

    NASA Astrophysics Data System (ADS)

    Huang, Qiang; Cun, Tangxiang; Zuo, Wenbin; Liu, Jianping

    2015-03-01

    We report the fabrication of hierarchically microstructured flower-like ZnO by a facile and single-step procedure involving poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPSA) assisted aqueous chemical method. The shapes and sizes can be controlled just by varying the concentrations of the water-soluble polymer. When a suitable PAMPAS concentration was utilized, uniform well-defined and mono-dispersed chrysanthemum-like ZnO microstructures based on nanorod building blocks were obtained. The formation mechanism of the hierarchical structure was presented. The structured studies using XRD, HRTEM and SAED reveal these ZnO nanorods are composed of a single phase nature with wurtzite structure and grow along with the c-axis. FTIR spectrum indicated the incorporation of a trace of PAMPSA into ZnO crystals. HRTEM, Raman and XPS analyses showed that the hierarchical ZnO microstructures contain high concentration of oxygen vacancies which enable them exhibiting a significant intense deep-level emission centered at green luminescence in its photoluminescence spectra. They also show enhanced photocatalytic efficiency in degradation of methylene blue. It is hoped that the present work may provide a simple method to fabricate ZnO hierarchical microstructures and a positive relationship among polar plane, oxygen vacancy and green emission.

  9. Synthesis of Polymer-Coated Magnetic Nanoparticles from Red Mud Waste for Enhanced Oil Recovery in Offshore Reservoirs

    NASA Astrophysics Data System (ADS)

    Nguyen, T. P.; Le, U. T. P.; Ngo, K. T.; Pham, K. D.; Dinh, L. X.

    2016-07-01

    Buried red mud waste from groundwater refineries can cause pollution. The aim of this paper is to utilize this mud for the synthesis of Fe3O4 magnetic nanoparticles (MNPs). Then, MNPs are encapsulated by a copolymer of methyl methacrylate and 2-acrylamido-2-methyl-1-propanesulfonate via oleic acid linker. MNPs are prepared by a controlled co-precipitation method in the presence of a dispersant and surface-modified agents to achieve a high hydrophobic or hydrophilic surface. Mini-emulsion polymerization was conducted to construct a core-shell structure with MNPs as core and the copolymer as shell. The core-shell structure of the obtained particles enables them to disperse well in brine and to stabilize at high-temperature environments. The chemical structures and morphology of this nanocomposite were investigated by Fourier transform infrared spectroscopy, transmission electron microscopy, and field emission scanning electron microscopy. The thermal stability of the nanocomposite was evaluated via a thermogravimetric analysis method for the solid state and an annealing experiment for the liquid state. The nanocomposite is about 14 nm, disperses well in brine and is thermally stable in the solid state. The blends of synthesized nanocomposite and carboxylate surfactant effectively reduced the interfacial tension between crude oil and brine, and remained thermally stable after 31 days annealed at 100°C. Therefore, a nanofluid of copolymer/magnetic nanocomposite can be applied as an enhanced oil recovery agent for harsh environments in offshore reservoirs.

  10. Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

    PubMed

    Ao, Mei; Domingue, Jada C; Khan, Nabihah; Javed, Fatima; Osmani, Kashif; Sarathy, Jayashree; Rao, Mrinalini C

    2016-06-01

    Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of

  11. An intelligent multicompartmental system based on thermo-sensitive starch microspheres for temperature-controlled release of drugs.

    PubMed

    Fundueanu, Gheorghe; Constantin, Marieta; Ascenzi, Paolo; Simionescu, Bogdan C

    2010-08-01

    An original multicompartmental drug delivery system based on encapsulation of "intelligent" starch microspheres was designed and developed. Starch microspheres with thermo-responsive properties and possessing strong anionic functional groups (-SO(3)H), capable to bind electrostatically drugs, has been prepared. Firstly, the thermo-responsive units based on copolymer of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) with a lower critical solution temperature around 36 degrees C, were grafted on preformed starch microspheres. Secondly, the strong anionic groups (-SO(3)H) were introduced by sequential grafting of 2-acrylamido-2-methyl-1-propanesulfonic acid on the remaining--OH groups of starch. The thermo-sensitive microspheres with sulfonic groups display a sharp phase transition around the human body temperature. They were complexed with the positively-charged metoclopramide (low molecular weight model drug) and then encapsulated in cellulose acetate butyrate microcapsules by an oil-in-water solvent evaporation method. The swelling and diffusion of encapsulated microspheres to the aqueous continuous phase is avoided because the temperature of aqueous phase is higher than volume phase transition temperature (VPTT) of microspheres. This multicompartmental device could develop the background of "smart" implantable drug delivery system for persons that work in dangerous cold places (builders, climbers). When the temperature of the human body decreases below the normal temperature (the threshold temperature could be tuned), the encapsulated microspheres swell extensively in contact with physiological fluids, break the microcapsules and a large amount of bioactive compounds is released, keeping the activity of the vital organs. In normal physiological conditions (above LCST), the microspheres slightly swell, fill up the microcavities of microcapsules, but do not break them and release the drug in microcompartments. These microcompartments become microreservoirs

  12. Conductive Polymeric Binder for Lithium-Ion Battery Anode

    NASA Astrophysics Data System (ADS)

    Gao, Tianxiang

    Tin (Sn) has a high-specific capacity (993 mAhg-1) as an anode material for Li-ion batteries. To overcome the poor cycling performance issue caused by its large volume expansion and pulverization during the charging and discharging process, many researchers put efforts into it. Most of the strategies are through nanostructured material design and introducing conductive polymer binders that serve as matrix of the active material in anode. This thesis aims for developing a novel method for preparing the anode to improve the capacity retention rate. This would require the anode to have high electrical conductivity, high ionic conductivity, and good mechanical properties, especially elasticity. Here the incorporation of a conducting polymer and a conductive hydrogel in Sn-based anodes using a one-step electrochemical deposition via a 3-electrode cell method is reported: the Sn particles and conductive component can be electrochemically synthesized and simultaneously deposited into a hybrid thin film onto the working electrode directly forming the anode. A well-defined three dimensional network structure consisting of Sn nanoparticles coated by conducting polymers is achieved. Such a conductive polymer-hydrogel network has multiple advantageous features: meshporous polymeric structure can offer the pathway for lithium ion transfer between the anode and electrolyte; the continuous electrically conductive polypyrrole network, with the electrostatic interaction with elastic, porous hydrogel, poly (2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile) (PAMPS) as both the crosslinker and doping anion for polypyrrole (PPy) can decrease the volume expansion by creating porous scaffold and softening the system itself. Furthermore, by increasing the amount of PAMPS and creating an interval can improve the cycling performance, resulting in improved capacity retention about 80% after 20 cycles, compared with only 54% of that of the control sample without PAMPS. The cycle

  13. Post-polymerization photografting on methacrylate-based monoliths for separation of intact proteins and protein digests with comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry.

    PubMed

    Vonk, Rudy J; Wouters, Sam; Barcaru, Andrei; Vivó-Truyols, Gabriel; Eeltink, Sebastiaan; de Koning, Leo J; Schoenmakers, Peter J

    2015-05-01

    Post-polymerization photografting is a versatile tool to alter the surface chemistry of organic-based monoliths so as to obtain desired stationary phase properties. In this study, 2-acrylamido-2-methyl-1-propanesulfonic acid was grafted to a hydrophobic poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith to create a strong cation exchange stationary phase. Both single-step and two-step photografting were addressed, and the effects of grafting conditions were assessed. An experimental design has been applied in an attempt to optimize three of the key parameters of the two-step photografting chemistry, i.e. the grafting time of the initiator, the monomer concentration and the monomer irradiation time. The photografted columns were implemented in a comprehensive two-dimensional column liquid chromatography ( (t) LC ×  (t) LC) workflow and applied for the separation of intact proteins and peptides. A baseline separation of 11 intact proteins was obtained within 20 min by implementing a gradient across a limited RP composition window in the second dimension. (t) LC ×  (t) LC with UV detection was used for the separation of cytochrome c digest, bovine serum insulin digest and a digest of a complex protein mixture. A semi-quantitative estimation of the occupation of separation space, the orthogonality, of the (t) LC ×  (t) LC system yielded 75%. The (t) LC ×  (t) LC setup was hyphenated to a high-resolution Fourier transform ion cyclotron resonance mass spectrometer instrument to identify the bovine serum insulin tryptic peptides and to demonstrate the compatibility with MS analysis.

  14. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

    PubMed

    Henin, N; Vincent, M F; Gruber, H E; Van den Berghe, G

    1995-04-01

    AMP-activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid synthesis, and 3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. AICAR (5-amino 4-imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10-fold rat liver AMP-activated protein kinase, with a half-maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50-500 microM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose-dependent inactivation of both acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. Addition of 50-500 microM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half-maximal inhibition was obtained at approximately 100 microM, and near-complete inhibition at 500 microM AICAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP-activated protein kinase.

  15. Ascorbic acid inhibits PMP22 expression by reducing cAMP levels.

    PubMed

    Kaya, Ferdinand; Belin, Sophie; Bourgeois, Patrice; Micaleff, Joelle; Blin, Olivier; Fontés, Michel

    2007-03-01

    Charcot-Marie-Tooth [CMT] syndrome is the most common hereditary peripheral neuropathy. CMT1A, which accounts for 50% of all CMT cases, usually results from triploidy of the PMP22 gene. Preclinical trials using an animal model show that disabled mice force-fed with high doses of ascorbic acid partially recover muscular strength after a few months of treatment, and suggest that high doses of ascorbic acid repress PMP22 expression. In this study, we demonstrated that ascorbic acid represses PMP22 gene expression by acting on intracellular cAMP levels and adenylate cyclase activity. This action is dose dependent and specific to ascorbic acid, since repression is not observed after treatment with other antioxidants. The new properties of ascorbic acid are discussed, along with the implications of these findings for CMT disease treatment.

  16. Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

    PubMed

    Bellotti, Marta; Salis, Annalisa; Grozio, Alessia; Damonte, Gianluca; Vigliarolo, Tiziana; Galatini, Andrea; Zocchi, Elena; Benatti, Umberto; Millo, Enrico

    2015-01-01

    The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.

  17. cAMP and forskolin decrease. gamma. -aminobutyric acid-gated chloride flux in rat brain synaptoneurosomes

    SciTech Connect

    Heuschneider, G.; Schwartz, R.D. )

    1989-04-01

    The effects of the cyclic nucleotide cAMP on {gamma}-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N{sup 6}, O{sup 2{prime}}-dibutyryladenosine 3{prime},5{prime}-cyclic monophosphate inhibited muscimol-induced {sup 36}Cl{sup {minus}} uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner. The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3{prime},5{prime}-cyclic monophosphate, 8-bromoadenosine 3{prime},5{prime}-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxanthine. The effect of endogenous cAMP accumulation on the {gamma}-aminobutyric acid-gated Cl{sup {minus}} channel was studied with forskolin, an activator of adenylate cyclase. Under identical conditions, in the intact synaptoneurosomes, forskolin inhibited muscimol-induced {sup 36}Cl{sup {minus}} uptake and generated cAMP with similar potencies. Surprisingly, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, also inhibited the muscimol response, suggesting that forskolin and its lipophilic derivatives may interact with the Cl{sup {minus}} channel directly. The data suggest that {gamma}-aminobutyric acid (GABA{sub A}) receptor function in brain can be regulated by cAMP-dependent phosphorylation.

  18. cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Cornejo Maciel, Fabiana; Castilla, Rocío; Duarte, Alejandra; Maloberti, Paula; Paz, Cristina; Podestá, Ernesto J

    2006-11-01

    We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.

  19. Radiation Effects on Cyclic AMP, Cyclic GMP, and Amino Acid Levels in the CSF of the Primate

    DTIC Science & Technology

    1980-11-07

    rradiation . An analysis of brain areas obtained by biopsy of irradiated animals showed significant decreases in only the cerebellar cyclic AMP and cyclic...GMP. No appreciablk ¢±anges were found in the CSF amino acid composition. Acc. !-,’r ror UV , . c" l. __ ..:j’od I7 .... ’U r~rd0 /or cpy I NCI

  20. Suppression of nephrin expression by TNF-alpha via interfering with the cAMP-retinoic acid receptor pathway.

    PubMed

    Saito, Yukinori; Okamura, Maro; Nakajima, Shotaro; Hayakawa, Kunihiro; Huang, Tao; Yao, Jian; Kitamura, Masanori

    2010-06-01

    Nephrin, a crucial component of the slit diaphragm, is downregulated in proteinuric glomerular diseases including glomerulonephritis. We previously reported that 1) expression of nephrin in cultured podocytes is reinforced by retinoic acid (RA) and 1,25-dihydroxyvitamin D(3), 2) these effects are mediated by retinoic acid receptor (RAR) and vitamin D receptor (VDR), and 3) basal and inducible expression of nephrin is downregulated by TNF-alpha. In the present investigation, we identified that TNF-alpha selectively represses activity of RAR but not VDR. To elucidate mechanisms underlying this observation, we tested involvement of downstream targets for TNF-alpha: nuclear factor-kappaB (NF-kappaB), mitogen-activated protein (MAP) kinases, phosphatidylinositol 3-kinase (PI3K)-Akt, and cAMP-protein kinase A (PKA). TNF-alpha caused activation of NF-kappaB, MAP kinases, and PI3K-Akt in podocytes, whereas blockade of these molecules did not affect inhibition of RAR by TNF-alpha. In contrast, TNF-alpha depressed activity of cAMP-PKA, and blockade of PKA inhibited basal and RA-induced activation of RAR. Furthermore, activity of RAR was significantly upregulated by cAMP, and the suppressive effect of TNF-alpha on RAR was reversed by cAMP-elevating agents. These results suggest that 1) expression of nephrin in podocytes is regulated by the cAMP-RAR pathway and 2) suppression of nephrin by TNF-alpha is caused, at least in part, through selective inhibition of this pathway.

  1. The ω-3 fatty acid eicosapentaenoic acid elicits cAMP generation in colonic epithelial cells via a “store-operated” mechanism

    PubMed Central

    Roy, Jessica; Lefkimmiatis, Konstantinos; Moyer, Mary Pat; Curci, Silvana

    2010-01-01

    Eicosapentaenoic acid (EPA) is an ω-3 polyunsaturated fatty acid abundant in fish oil that exerts a wide spectrum of documented beneficial health effects in humans. Because dietary interventions are relatively inexpensive and are widely assumed to be safe, they have broad public appeal. Their endorsement can potentially have a major impact on human health, but hard mechanistic evidence that specifies how these derivatives work at the cellular level is limited. EPA (50 μM) caused a small elevation of cytoplasmic Ca2+ concentration ([Ca2+]) in intact NCM460 human colonic epithelial cells as measured by fura 2 and a profound drop of [Ca2+] within the endoplasmic reticulum (ER) of permeabilized cells as monitored by compartmentalized mag-fura 2. Total internal reflection fluorescence microscopy showed that this loss of ER store [Ca2+] led to translocation of the ER-resident transmembrane Ca2+ sensor STIM1. Using sensitive FRET-based sensors for cAMP in single cells, we further found that EPA caused a substantial increase in cellular cAMP concentration, a large fraction of which was dependent on the drop in ER [Ca2+], but independent of cytosolic Ca2+. An additional component of the EPA-induced cAMP signal was sensitive to the phosphodiesterase inhibitor isobutyl methylxanthine. We conclude that EPA slowly releases ER Ca2+ stores, resulting in the generation of cAMP. The elevated cAMP is apparently independent of classical G protein-coupled receptor activation and is likely the consequence of a newly described “store-operated” cAMP signaling pathway that is mediated by STIM1. PMID:20576916

  2. Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells.

    PubMed

    Jung, YunJae

    2015-12-01

    Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

  3. Downstream molecular events in the altered profiles of lysophosphatidic acid-induced cAMP in senescent human diploid fibroblasts.

    PubMed

    Jang, Ik Soon; Rhim, Ji Heon; Park, Sang Chul; Yeo, Eui Ju

    2006-04-30

    Lysophosphatidic acid (LPA) is a phospholipid growth factor that acts through G-protein-coupled receptors. Previously, we demonstrated an altered profile of LPA-dependent cAMP content during the aging process of human diploid fibroblasts (HDFs). In attempts to define the molecular events associated with the age-dependent changes in cAMP profiles, we determined the protein kinase A (PKA) activity, phosphorylation of cAMP-response element binding protein (CREB), and the protein expression of CRE-regulatory genes, c-fos and COX-2 in young and senescent HDFs. We observed in senescent cells, an increase in mRNA levels of the catalytic subunit a of PKA and of the major regulatory subunit Ialpha. Senescence-associated increase of cAMP after LPA treatment correlated well with increased CREB phosphorylation accompanying activation of PKA in senescent cells. In senescent cells, after LPA treatment, the expression of c-fos and COX-2 decreased initially, followed by an increase. In young HDFs, CREB phosphorylation decreased following LPA treatment, and both c-fos and COX-2 protein levels increased rapidly. CRE-luciferase assay revealed higher basal CRE-dependent gene expression in young HDFs compared to senescent HDFs. However, LPA-dependent slope of luciferase increased more rapidly in senescent cells than in young cells, presumably due to an increase of LPA-induced CREB phosphorylation. CRE-dependent luciferase activation was abrogated in the presence of inhibitors of PKC, MEK1, p38MAPK, and PKA, in both young and senescent HDFs. We conclude that these kinase are coactivators of the expression of CRE-responsive genes in LPA-induced HDFs and that their changed activities during the aging process contribute to the final expression level of CRE-responsive genes.

  4. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    PubMed

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin.

  5. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells

    PubMed Central

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin. PMID:27348124

  6. [Effect of aluminium and cAMP on acid phosphatase from the apoplast of barley and maize root cells].

    PubMed

    Fedorovskaia, M D; Tikhaia, N I

    2003-01-01

    Acid phosphatase activity inhibited by 1 mM sodium molybdate was detected at the surface of barley seedling roots and in the cell wall fraction isolated from barley and maize seedling roots. This enzyme hydrolyzed NPP, GP, and PPi at low pH (4.0 and below). NPP hydrolysis was stimulated by magnesium (but not calcium or manganese) ions, while PPi hydrolysis was independent of the presence of bivalent ions. The activity of phosphatase localized in the cell walls of the both crops increased in the presence of 100 microM AlCl3 or CuCl2. Stimulation of NPP hydrolysis by micromolar concentrations of aluminium and copper as well as by millimolar concentrations of magnesium decreased in the presence of 25 microM cAMP. This agrees with the previous data on the enzyme localized at the outer side of the properly oriented vesicles in the microscomal fraction of plasmalemma. The role of the root extracellular acid phosphatase loosely associated with various apoplast structures in plant adaptation to toxic effect of aluminium in the acidic soils as well as possible control of this process by cAMP secretion to the apoplast are discussed.

  7. Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5'-AMP with hydrophobic amino acids

    NASA Technical Reports Server (NTRS)

    Lacey, J. C. Jr; Wickramasinghe, N. S.; Sabatini, R. S.

    1992-01-01

    We have studied the chemistry of aminoacyl AMP to model reactions at the 3' terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5'-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The beta-branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the gamma-branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with adenine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.

  8. Phosphorylation of CREB and mechanical hyperalgesia is reversed by blockade of the cAMP pathway in a time-dependent manner after repeated intramuscular acid injections.

    PubMed

    Hoeger-Bement, Marie K; Sluka, Kathleen A

    2003-07-02

    Spinal activation of the cAMP pathway produces mechanical hyperalgesia, sensitizes nociceptive spinal neurons, and phosphorylates the transcription factor cAMP-responsive element binding protein (CREB), which initiates gene transcription. This study examined the role of the cAMP pathway in a model of chronic muscle pain by assessing associated behavioral changes and phosphorylation of CREB. Bilateral mechanical hyperalgesia of the paw was induced by administering two injections of acidic saline, 5 d apart, into the gastrocnemius muscle of male Sprague Dawley rats. Interestingly, the increases in immunoreactivity for CREB and phosphorylated CREB (p-CREB) in the spinal dorsal horn occur 24 hr, but not 1 week, after the second injection of acidic saline compared with pH 7.2 intramuscular injections. Spinal blockade of adenylate cyclase prevents the expected increase in p-CREB that occurs after intramuscular acid injection. The reversal of mechanical hyperalgesia by adenylate cyclase or protein kinase A inhibitors spinally follows a similar pattern with reversal at 24 hr, but not 1 week, compared with the vehicle controls. The p-CREB immunoreactivity in the superficial dorsal horn correlates with the mechanical withdrawal threshold such that increases in p-CREB are associated with decreases in threshold. Therefore, activation of the cAMP pathway in the spinal cord phosphorylates CREB and produces mechanical hyperalgesia associated with intramuscular acid injections. The mechanical hyperalgesia and phosphorylation of CREB depend on early activation of the cAMP pathway during the first 24 hr but are independent of the cAMP pathway by 1 week after intramuscular injection of acid.

  9. Folic acid supplementation during high-fat diet feeding restores AMPK activation via an AMP-LKB1-dependent mechanism.

    PubMed

    Sid, Victoria; Wu, Nan; Sarna, Lindsei K; Siow, Yaw L; House, James D; O, Karmin

    2015-11-15

    AMPK is an endogenous energy sensor that regulates lipid and carbohydrate metabolism. Nonalcoholic fatty liver disease (NAFLD) is regarded as a hepatic manifestation of metabolic syndrome with impaired lipid and glucose metabolism and increased oxidative stress. Our recent study showed that folic acid supplementation attenuated hepatic oxidative stress and lipid accumulation in high-fat diet-fed mice. The aim of the present study was to investigate the effect of folic acid on hepatic AMPK during high-fat diet feeding and the mechanisms involved. Male C57BL/6J mice were fed a control diet (10% kcal fat), a high-fat diet (60% kcal fat), or a high-fat diet supplemented with folic acid (26 mg/kg diet) for 5 wk. Mice fed a high-fat diet exhibited hyperglycemia, hepatic cholesterol accumulation, and reduced hepatic AMPK phosphorylation. Folic acid supplementation restored AMPK phosphorylation (activation) and reduced blood glucose and hepatic cholesterol levels. Activation of AMPK by folic acid was mediated through an elevation of its allosteric activator AMP and activation of its upstream kinase, namely, liver kinase B1 (LKB1) in the liver. Consistent with in vivo findings, 5-methyltetrahydrofolate (bioactive form of folate) restored phosphorylation (activation) of both AMPK and LKB1 in palmitic acid-treated HepG2 cells. Activation of AMPK by folic acid might be responsible for AMPK-dependent phosphorylation of HMG-CoA reductase, leading to reduced hepatic cholesterol synthesis during high-fat diet feeding. These results suggest that folic acid supplementation may improve cholesterol and glucose metabolism by restoration of AMPK activation in the liver.

  10. G protein-coupled receptor Gpr4 senses amino acids and activates the cAMP-PKA pathway in Cryptococcus neoformans.

    PubMed

    Xue, Chaoyang; Bahn, Yong-Sun; Cox, Gary M; Heitman, Joseph

    2006-02-01

    The Galpha protein Gpa1 governs the cAMP-PKA signaling pathway and plays a central role in virulence and differentiation in the human fungal pathogen Cryptococcus neoformans, but the signals and receptors that trigger this pathway were unknown. We identified seven putative proteins that share identity with known G protein-coupled receptors (GPCRs). One protein, Gpr4, shares limited sequence identity with the Dictyostelium discoideum cAMP receptor cAR1 and the Aspergillus nidulans GPCR protein GprH and also shares structural similarity with the Saccharomyces cerevisiae receptor Gpr1. gpr4 mutants exhibited reduced capsule production and mating defects, similar to gpa1 mutants, and exogenous cAMP suppressed both gpr4 mutant phenotypes. Epistasis analysis provides further evidence that Gpr4 functions upstream of the Galpha subunit Gpa1. Gpr4-Gpr4 homomeric interactions were observed in the yeast two-hybrid assay, and Gpr4 was shown to physically interact with Gpa1 in the split-ubiquitin system. A Gpr4::DsRED fusion protein was localized to the plasma membrane and methionine was found to trigger receptor internalization. The analysis of intracellular cAMP levels showed that gpr4 mutants still respond to glucose but not to certain amino acids, such as methionine. Amino acids might serve as ligands for Gpr4 and could contribute to engage the cAMP-PKA pathway. Activation of the cAMP-PKA pathway by glucose and amino acids represents a nutrient coincidence detection system shared in other pathogenic fungi.

  11. The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation.

    PubMed

    Gualdoni, Guido A; Mayer, Katharina A; Göschl, Lisa; Boucheron, Nicole; Ellmeier, Wilfried; Zlabinger, Gerhard J

    2016-11-01

    T cells must tightly regulate their metabolic processes to cope with varying bioenergetic demands depending on their state of differentiation. The metabolic sensor AMPK is activated in states of low energy supply and modulates cellular metabolism toward a catabolic state. Although this enzyme is known to be particularly active in regulatory T (Treg) cells, its impact on T helper (Th)-cell differentiation is poorly understood. We investigated the impact of several AMPK activators on Treg-cell differentiation and found that the direct activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide), but not the indirect activators metformin and 2-deoxyglucose, strongly enhanced Treg-cell induction by specifically enhancing Treg-cell expansion. Conversely, Th17 generation was impaired by the agent. Further investigation of the metabolic background of our observations revealed that AICAR enhanced both cellular mitochondrogenesis and fatty acid uptake. Consistently, increased Treg induction was entirely reversible on inhibition of fatty acid oxidation, thus confirming the dependence of AICAR's effects on metabolic pathways alterations. Translating our findings to an in vivo model, we found that the substance enhanced Treg cell generation on IL-2 complex-induced immune stimulation. We provide a previously unrecognized insight into the delicate interplay between immune cell function and metabolism and delineate a potential novel strategy for metabolism-targeting immunotherapy.-Gualdoni, G. A., Mayer, K. A., Göschl, L., Boucheron, N., Ellmeier, W., Zlabinger, G. J. The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation.

  12. Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells

    PubMed Central

    Salinthone, Sonemany; Schillace, Robynn V.; Marracci, Gail H.; Bourdette, Dennis N.; Carr, Daniel W.

    2008-01-01

    The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells in a dose-dependent manner. Studies using pharmacological inhibitors and receptor transfection experiments indicate that LA stimulates cAMP production via activation of the EP2 and EP4 prostanoid receptors and adenylyl cyclase. In addition, LA suppressed interleukin (IL)-12/IL-18 induced IFNγ secretion and cytotoxicity in NK cells. These novel findings suggest that LA may inhibit NK cell function via the cAMP signaling pathway. PMID:18562016

  13. Lipoic acid stimulates cAMP production via the EP2 and EP4 prostanoid receptors and inhibits IFN gamma synthesis and cellular cytotoxicity in NK cells.

    PubMed

    Salinthone, Sonemany; Schillace, Robynn V; Marracci, Gail H; Bourdette, Dennis N; Carr, Daniel W

    2008-08-13

    The antioxidant lipoic acid (LA) treats and prevents the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). In an effort to understand the therapeutic potential of LA in MS, we sought to define the cellular mechanisms that mediate the effects of LA on human natural killer (NK) cells, which are important in innate immunity as the first line of defense against invading pathogens and tumor cells. We discovered that LA stimulates cAMP production in NK cells in a dose-dependent manner. Studies using pharmacological inhibitors and receptor transfection experiments indicate that LA stimulates cAMP production via activation of the EP2 and EP4 prostanoid receptors and adenylyl cyclase. In addition, LA suppressed interleukin (IL)-12/IL-18 induced IFNgamma secretion and cytotoxicity in NK cells. These novel findings suggest that LA may inhibit NK cell function via the cAMP signaling pathway.

  14. Nonsteroidal anti-inflammatory drug flufenamic acid is a potent activator of AMP-activated protein kinase.

    PubMed

    Chi, Yuan; Li, Kai; Yan, Qiaojing; Koizumi, Schuichi; Shi, Liye; Takahashi, Shuhei; Zhu, Ying; Matsue, Hiroyuki; Takeda, Masayuki; Kitamura, Masanori; Yao, Jian

    2011-10-01

    Flufenamic acid (FFA) is a nonsteroidal anti-inflammatory drug (NSAID). It has anti-inflammatory and antipyretic properties. In addition, it modulates multiple channel activities. The mechanisms underlying the pharmacological actions of FFA are presently unclear. Given that AMP-activated protein kinase (AMPK) has both anti-inflammatory and channel-regulating functions, we examined whether FFA induces AMPK activation. 1) Exposure of several different types of cells to FFA resulted in an elevation of AMPKα phosphorylation at Thr172. This effect of FFA was reproduced by functionally and structurally similar mefenamic acid, tolfenamic acid, niflumic acid, and meclofenamic acid. 2) FFA-induced activation of AMPK was largely abolished by the treatment of cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (an intracellular Ca(2+) chelator) or depletion of extracellular Ca(2+), whereas it was mimicked by stimulation of cells with the Ca(2+) ionophore 5-(methylamino)-2-({(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl}methyl)-1,3-benzoxazole-4-carboxylic acid (A23187) or ionomycin. 3) FFA triggered a rise in intracellular Ca(2+), which was abolished by cyclosporine, a blocker of mitochondrial permeability transition pore. Cyclosporine also abolished FFA-induced activation of AMPK. 4) Inhibition of Ca(2+)/calmodulin-dependent kinase kinase β (CaMKKβ) with 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609) or down-regulation of CaMKKβ with short interfering RNA largely abrogated FFA-induced activation of AMPK. 5) FFA significantly suppressed nuclear factor-κB activity and inducible nitric-oxide synthase expression triggered by interleukin-1β and tumor necrosis factor α. This suppression was also largely abrogated by STO-609. Taken together, we conclude that FFA induces AMPK activation through the Ca(2+)-CaMKKβ pathway

  15. Highly efficient peptide formation from N-acetylaminoacyl-AMP anhydride and free amino acid

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1983-01-01

    The kinetics of formation of the N-blocked dipeptide, N-acetylglycylglycine, from N-acetylglycyl adenylate anhydride and glycine in aqueous solution at 25 C, and at various PH's are reported. The reaction is of interest in that over a physiologically relevant pH range (6-8), peptide synthesis proceeds more rapidly than hydrolysis, even at those pH's at which this compound becomes increasingly susceptible to base-catalyzed hydrolysis. Under similar conditions, the corresponding unblocked aminoacyl adenylate anhydrides are considerably more unstable, and undergo appreciable hydrlysis in the presence of free amino acid. Because N-blocked aminoacyl adenylate anhydrides serve as model compounds of peptidyl adenylate anhydrides, these results suggest that primitive amino acid polymerization systems may have operated by cyclic reactivation of the peptidyl carboxyl group, rather than that of the incoming amino acid.

  16. AMP kinase activation with AICAR further increases fatty acid oxidation and blunts triacylglycerol hydrolysis in contracting rat soleus muscle.

    PubMed

    Smith, Angela C; Bruce, Clinton R; Dyck, David J

    2005-06-01

    Muscle contraction increases glucose uptake and fatty acid (FA) metabolism in isolated rat skeletal muscle, due at least in part to an increase in AMP-activated kinase activity (AMPK). However, the extent to which AMPK plays a role in the regulation of substrate utilization during contraction is not fully understood. We examined the acute effects of 5-aminoimidazole-4-carboxamide riboside (AICAR; 2 mm), a pharmacological activator of AMPK, on FA metabolism and glucose oxidation during high intensity tetanic contraction in isolated rat soleus muscle strips. Muscle strips were exposed to two different FA concentrations (low fatty acid, LFA, 0.2 mm; high fatty acid, HFA, 1 mm) to examine the role that FA availability may play in both exogenous and endogenous FA metabolism with contraction and AICAR. Synergistic increases in AMPK alpha2 activity (+45%; P<0.05) were observed after 30 min of contraction with AICAR, which further increased exogenous FA oxidation (LFA: +71%, P<0.05; HFA: +46%, P<0.05) regardless of FA availability. While there were no changes in triacylglycerol (TAG) esterification, AICAR did increase the ratio of FA partitioned to oxidation relative to TAG esterification (LFA: +65%, P<0.05). AICAR significantly blunted endogenous TAG hydrolysis (LFA: -294%, P<0.001; HFA: -117%, P<0.05), but had no effect on endogenous oxidation rates, suggesting a better matching between TAG hydrolysis and subsequent oxidative needs of the muscle. There was no effect of AICAR on the already elevated rates of glucose oxidation during contraction. These results suggest that FA metabolism is very sensitive to AMPK alpha2 stimulation during contraction.

  17. Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase.

    PubMed

    Kuby, S A; Palmieri, R H; Frischat, A; Fischer, A H; Wu, L H; Maland, L; Manship, M

    1984-05-22

    The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Elements of the glucocorticoid and retinoic acid response units are involved in cAMP-mediated expression of the PEPCK gene.

    PubMed

    Waltner-Law, Mary; Duong, David T; Daniels, Marc C; Herzog, Birger; Wang, Xiaohui L; Prasad, Ratna; Granner, Daryl K

    2003-03-21

    Although many genes are regulated by the concerted action of several hormones, hormonal signaling to gene promoters has generally been studied one hormone at a time. The phosphoenolpyruvate carboxykinase (PEPCK) gene is a case in point. Transcription of this gene is induced by glucagon (acting by the second messenger, cAMP), glucocorticoids, and retinoic acid, and it is dominantly repressed by insulin. These hormonal responses require the presence of different hormone response units (HRUs), which consist of constellations of DNA elements and associated transcription factors. These include the glucocorticoid response unit (GRU), cAMP response unit (CRU), retinoic acid response unit (RARU), and the insulin response unit. HRUs are known to have functional overlap. In particular, the cAMP response element of the CRU is also a component of the GRU. The purpose of this study was to determine whether known GRU or RARU elements or transcription factors function as components of the CRU. We show here that the glucocorticoid accessory factor binding site 1 and glucocorticoid accessory factor binding site 3 elements, which are components of both the GRU and RARU, are an important part of the CRU. Furthermore, we find that the transcription factor, chicken ovalbumin upstream promoter-transcription factor, and two coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, participate in both the cAMP and glucocorticoid responses. This provides a further illustration of how the PEPCK gene promoter integrates different hormone responses through overlapping HRUs that utilize some of the same transcription factors and coactivators.

  19. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model.

    PubMed

    Drab, M; Haller, H; Bychkov, R; Erdmann, B; Lindschau, C; Haase, H; Morano, I; Luft, F C; Wobus, A M

    1997-09-01

    Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.

  20. Uric acid-dependent inhibition of AMP kinase induces hepatic glucose production in diabetes and starvation: evolutionary implications of the uricase loss in hominids.

    PubMed

    Cicerchi, Christina; Li, Nanxing; Kratzer, James; Garcia, Gabriela; Roncal-Jimenez, Carlos A; Tanabe, Katsuyuki; Hunter, Brandi; Rivard, Christopher J; Sautin, Yuri Y; Gaucher, Eric A; Johnson, Richard J; Lanaspa, Miguel A

    2014-08-01

    Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.

  1. Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes.

    PubMed Central

    Luiken, J J F P; Willems, J; Coort, S L M; Coumans, W A; Bonen, A; Van Der Vusse, G J; Glatz, J F C

    2002-01-01

    Recently, we established that cellular contractions increase long-chain fatty-acid (FA) uptake by cardiac myocytes. This increase is dependent on the transport function of an 88 kDa membrane FA transporter, FA translocase (FAT/CD36), and, in analogy to skeletal muscle, is likely to involve its translocation from an intracellular pool to the sarcolemma. In the present study, we investigated whether cAMP-dependent signalling is involved in this translocation process. Isoproterenol, dibutyryl-cAMP and the phosphodiesterase (PDE) inhibitor, amrinone, which markedly raised the intracellular cAMP level, did not affect cellular FA uptake, but influenced the fate of intracellular FAs by directing these to mitochondrial oxidation in electrostimulated cardiac myocytes. The PDE inhibitors 3-isobutyl-1-methylxanthine, milrinone and dipyridamole each significantly stimulated FA uptake as well as intracellular cAMP levels, but these effects were quantitatively unrelated. The stimulatory effects of these PDE inhibitors were antagonized by sulpho- N -succinimidylpalmitate, indicating the involvement of FAT/CD36, albeit that the different PDE inhibitors use different molecular mechanisms to stimulate FAT/CD36-mediated FA uptake. Notably, 3-isobutyl-1-methylxanthine and milrinone increased the intrinsic activity of FAT/CD36, possibly through its covalent modification, and dipyridamole induces translocation of FAT/CD36 to the sarcolemma. Elevation of intracellular cGMP, but not of cAMP, by the PDE inhibitor zaprinast did not have any effect on FA uptake and metabolism by cardiac myocytes. The stimulatory effects of PDE inhibitors on cardiac FA uptake should be considered when applying these agents in clinical medicine. PMID:12093365

  2. Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-Activated Protein Kinase Alpha In vivo and In vitro

    PubMed Central

    Lim, Hara; Park, Jinbong; Kim, Hye-Lin; Kang, JongWook; Jeong, Mi-Young; Youn, Dong-Hyun; Jung, Yunu; Kim, Yong-Il; Kim, Hyun-Ju; Ahn, Kwang Seok; Kim, Su-Jin; Choe, Seong-Kyu; Hong, Seung-Heon; Um, Jae-Young

    2016-01-01

    Chrysophanic acid (CA) is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD)-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK) inhibitor, the action of CA on AMPKα was nullified in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases, we suggest the new potential of CA as an anti-obese pharmacotherapy. PMID:28008317

  3. Chrysophanic Acid Suppresses Adipogenesis and Induces Thermogenesis by Activating AMP-Activated Protein Kinase Alpha In vivo and In vitro.

    PubMed

    Lim, Hara; Park, Jinbong; Kim, Hye-Lin; Kang, JongWook; Jeong, Mi-Young; Youn, Dong-Hyun; Jung, Yunu; Kim, Yong-Il; Kim, Hyun-Ju; Ahn, Kwang Seok; Kim, Su-Jin; Choe, Seong-Kyu; Hong, Seung-Heon; Um, Jae-Young

    2016-01-01

    Chrysophanic acid (CA) is a member of the anthraquinone family abundant in rhubarb, a widely used herb for obesity treatment in Traditional Korean Medicine. Though several studies have indicated numerous features of CA, no study has yet reported the effect of CA on obesity. In this study, we tried to identify the anti-obesity effects of CA. By using 3T3-L1 adipocytes and primary cultured brown adipocytes as in vitro models, high-fat diet (HFD)-induced obese mice, and zebrafish as in vivo models, we determined the anti-obesity effects of CA. CA reduced weight gain in HFD-induced obese mice. They also decreased lipid accumulation and the expressions of adipogenesis factors including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in 3T3-L1 adipocytes. In addition, uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), the brown fat specific thermogenic genes, were up-regulated in brown adipocytes by CA treatment. Furthermore, when co-treated with Compound C, the AMP-activated protein kinase (AMPK) inhibitor, the action of CA on AMPKα was nullified in both types of adipocytes, indicating the multi-controlling effect of CA was partially via the AMPKα pathway. Given all together, these results indicate that CA can ameliorate obesity by controlling the adipogenic and thermogenic pathway at the same time. On these bases, we suggest the new potential of CA as an anti-obese pharmacotherapy.

  4. Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP.

    PubMed

    Chao, C C; Sun, N K; Lin-Chao, S

    1993-02-15

    A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

  5. An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase.

    PubMed Central

    Cheng, H C; van Patten, S M; Smith, A J; Walsh, D A

    1985-01-01

    Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein-substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme. PMID:3000357

  6. Niflumic acid-sensitive ion channels play an important role in the induction of glucose-stimulated insulin secretion by cyclic AMP in mice

    PubMed Central

    Fujimoto, W.; Miki, T.; Ogura, T.; Zhang, M.; Seino, Y.; Satin, L. S.; Nakaya, H.

    2015-01-01

    Aims/hypothesis We have previously reported that glucose-stimulated insulin secretion (GSIS) is induced by glucagon-like peptide-1 (GLP-1) in mice lacking ATP-sensitive K+ (KATP) channels (Kir6.2−/− mice [up-to-date symbol for Kir6.2 gene is Kcnj11]), in which glucose alone does not trigger insulin secretion. This study aimed to clarify the mechanism involved in the induction of GSIS by GLP-1. Methods Pancreas perfusion experiments were performed using wild-type (Kir6.2+/+) or Kir6.2−/− mice. Glucose concentrations were either changed abruptly from 2.8 to 16.7 mmol/l or increased stepwise (1.4 mmol/l per step) from 2.8 to 12.5 mmol/l. Electrophysiological experiments were performed using pancreatic beta cells isolated from Kir6.2−/− mice or clonal pancreatic beta cells (MIN6 cells) after pharmacologically inhibiting their KATP channels with glibenclamide. Results The combination of cyclic AMP plus 16.7 mmol/l glucose evoked insulin secretion in Kir6.2−/− pancreases where glucose alone was ineffective as a secretagogue. The secretion was blocked by the application of niflumic acid. In KATP channel-inactivated MIN6 cells, niflumic acid similarly inhibited the membrane depolarisation caused by cAMP plus glucose. Surprisingly, stepwise increases of glucose concentration triggered insulin secretion only in the presence of cAMP or GLP-1 in Kir6.2+/+, as in Kir6.2−/− pancreases. Conclusions/interpretation Niflumic acid-sensitive ion channels participate in the induction of GSIS by cyclic AMP in Kir6.2−/− beta cells. Cyclic AMP thus not only acts as a potentiator of insulin secretion, but appears to be permissive for GSIS via novel, niflumic acid-sensitive ion channels. This mechanism may be physiologically important for triggering insulin secretion when the plasma glucose concentration increases gradually rather than abruptly. PMID:19266181

  7. Modulation of cAMP levels by high-fat diet and curcumin and regulatory effects on CD36/FAT scavenger receptor/fatty acids transporter gene expression.

    PubMed

    Zingg, Jean-Marc; Hasan, Syeda T; Nakagawa, Kiyotaka; Canepa, Elisa; Ricciarelli, Roberta; Villacorta, Luis; Azzi, Angelo; Meydani, Mohsen

    2017-01-02

    Curcumin, a polyphenol from turmeric (Curcuma longa), reduces inflammation, atherosclerosis, and obesity in several animal studies. In Ldlr(-/-) mice fed a high-fat diet (HFD), curcumin reduces plasma lipid levels, therefore contributing to a lower accumulation of lipids and to reduced expression of fatty acid transport proteins (CD36/FAT, FABP4/aP2) in peritoneal macrophages. In this study, we analyzed the molecular mechanisms by which curcumin (500, 1000, 1500 mg/kg diet, for 4 months) may influence plasma and tissue lipid levels in Ldlr(-/-) mice fed an HFD. In liver, HFD significantly suppressed cAMP levels, and curcumin restored almost normal levels. Similar trends were observed in adipose tissues, but not in brain, skeletal muscle, spleen, and kidney. Treatment with curcumin increased phosphorylation of CREB in liver, what may play a role in regulatory effects of curcumin in lipid homeostasis. In cell lines, curcumin increased the level of cAMP, activated the transcription factor CREB and the human CD36 promoter via a sequence containing a consensus CREB response element. Regulatory effects of HFD and Cur on gene expression were observed in liver, less in skeletal muscle and not in brain. Since the cAMP/protein kinase A (PKA)/CREB pathway plays an important role in lipid homeostasis, energy expenditure, and thermogenesis by increasing lipolysis and fatty acid β-oxidation, an increase in cAMP levels induced by curcumin may contribute to its hypolipidemic and anti-atherosclerotic effects. © 2016 BioFactors, 43(1):42-53, 2017.

  8. Cyclic AMP regulation of arachidonic acid (AA) release and phospholipid metabolism in human monocytes: modulation by intracellular calcium

    SciTech Connect

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

    1986-05-01

    Stimulation of inflammatory cells by specific ligands results in activation of phospholipase(s) and production of oxygenation products of AA. The authors have employed (/sup 3/H)AA labeled monocytes to examine the involvement of cAMP in regulating phospholipase activity as measured by percent of incorporated (/sup 3/H)AA released and TLC analysis of (/sup 3/H)AA cellular lipids. Maximum release of radiolabel (31 +/- 5%) occurred upon challenge with the calcium ionophore A23187/sup -/ (10..mu..M), while FMLP (1..mu..M) yielded 15 +/- 1% and untreated cells 8 +/- 1%. Pretreatment of monocytes with isobutyl methyl xanthine/sup -/(IBMX) or dibutyrl cyclic AMP (d-cAMP) inhibited FMLP stimulated release with IC/sub 50/'s of 2.5 x 10/sup -5/M and 8 x 10/sup -5/M respectively. Exposure of monocytes to maximal levels of IBMX (5 x 10/sup -4/M) or d-cAMP (10/sup -3/M) also reduced release from controls by 40%, while A23187 induced release was uneffected by either. Examination of (/sup 3/H) AA labeled phospholipids showed that phosphatidylcholine (PC) and phosphatidylinositol were the major pools labeled and that stimulation by FMLP or A23187 appeared to deplete the PC pool exclusively. Prior exposure to IBMX or d-cAMP inhibited the loss from the PC pool only in untreated or FMLP stimulated cells. The data suggests that a phospholipase A/sub 2/ activity, directly primarily towards PC, is regulated by cAMP possibly by inhibiting receptor mediated increases in intracellular calcium levels.

  9. Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells

    PubMed Central

    Wang, Jian-Fa; Fu, Shou-Peng; Li, Su-Nan; Hu, Zhong-Ming; Xue, Wen-Jing; Li, Zhi-Qiang; Huang, Bing-Xu; Lv, Qing-Kang; Liu, Ju-Xiong; Wang, Wei

    2013-01-01

    Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake. PMID:24177567

  10. The effects of forskolin and rolipram on cAMP, cGMP and free fatty acid levels in diet induced obesity.

    PubMed

    Doseyici, S; Mehmetoglu, I; Toker, A; Yerlikaya, F H; Erbay, E

    2014-07-01

    Obesity is a major health problem. We investigated the effects of forskolin and rolipram in the diet of animals in which obesity had been induced. We used 50 female albino Wistar rats that were assigned randomly into five groups as follows: group 1, control; group 2, high fat diet; group 3, high fat diet + forskolin; group 4, high fat diet + rolipram; and group 5, high fat diet + rolipram + forskolin. The rats were fed for 10 weeks and rolipram and forskolin were administered during last two weeks. The animals were sacrificed and blood samples were obtained. Serum cAMP, cGMP and free fatty acids (FFA) levels were measured using ELISA assays. We also measured weight gain during the 10 week period. cAMP and FFA levels of groups 3, 4 and 5 were significantly higher than those of groups 1 and 2. We found no significant differences in serum cGMP levels among the groups. The weight gain in groups 3, 4 and 5 was significantly less than for group 2. We also found that the weight gain in group 5 was significantly less than in groups 3 and 4. We found that both forskolin and rolipram stimulated lipolysis and inhibited body weight increase by increasing cAMP levels. Also, combination therapy using the two agents may be more effective in preventing diet induced obesity than either agent alone. We found also that these agents did not effect cellular cGMP levels in diet induced obesity.

  11. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    PubMed

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  12. p-Coumaric acid modulates glucose and lipid metabolism via AMP-activated protein kinase in L6 skeletal muscle cells.

    PubMed

    Yoon, Seon-A; Kang, Seong-Il; Shin, Hye-Sun; Kang, Seung-Woo; Kim, Jeong-Hwan; Ko, Hee-Chul; Kim, Se-Jae

    2013-03-22

    p-Coumaric acid (3-[4-hydroxyphenyl]-2-propenoic acid) is a ubiquitous plant metabolite with antioxidant, anti-inflammatory, and anticancer properties. In this study, we examined whether p-coumaric acid modulates glucose and lipid metabolism via AMP-activated protein kinase (AMPK) in L6 skeletal muscle cells. p-Coumaric acid increased the phosphorylation of AMPK in a dose-dependent manner in differentiated L6 skeletal muscle cells. It also increased the phosphorylation of acetyl-CoA carboxylase (ACC) and the expression of CPT-1 mRNA and PPARα, suggesting that it promotes the β-oxidation of fatty acids. Also, it suppressed oleic acid-induced triglyceride accumulation, and enhanced 2-NBDG uptake in differentiated L6 muscle cells. Pretreatment with compound C inhibited AMPK activation, reduced ACC phosphorylation and 2-NBDG uptake, and increased triglyceride accumulation. However, p-coumaric acid counterbalanced the inhibitory effects of compound C. Taken together, these results suggest that p-coumaric acid modulates glucose and lipid metabolism via AMPK activation in L6 skeletal muscle cells and that it has potentially beneficial effects in improving or treating metabolic disorders.

  13. Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells

    PubMed Central

    Huang, Hao; Hong, Qian; Tan, Hong-ling; Xiao, Cheng-rong; Gao, Yue

    2016-01-01

    Aim: Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells. Methods: PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA. Results: Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10–40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2. Conclusion: The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS

  14. Involvement of fatty acid-CoA ligase 4 in hepatocellular carcinoma growth: Roles of cyclic AMP and p38 mitogen-activated protein kinase

    PubMed Central

    Liang, Yu-Chih; Wu, Chih-Hsiung; Chu, Jan-Show; Wang, Chung-Kwe; Hung, Ling-Fang; Wang, Ying-Jan; Ho, Yuan-Soon; Chang, Jan-Gowth; Lin, Shyr-Yi

    2005-01-01

    AIM: Fatty acid-CoA ligase 4 (FACL4) is an arachidonate-preferring enzyme which has been shown to be up-regulated in human colon cancer tissues and implicated in the colon tumorigenesis. The purpose of this study was to investigate the role of FACL4 in the human hepatocellular carcinoma (HCC) tumorigenesis and the specific signal pathways involved in this process. METHODS: We investigated the expression and regulation of FACL4 in HCC, adjacent non-tumorous liver tissues, and cell lines. RESULTS: In HCC patients, we demonstrated that FACL4 gene expression was markedly elevated in the cancerous tissues than in the adjacent non-cancerous liver tissues. In addition, several human hepatoma cell lines, including Hep3B and HepG2, expressed high levels of FACL4. Stable overex-pression of FACL4 knockdown plasmids (small interfering RNA, siRNA) to Hep3B cells significantly decreased FACL4 expression and subsequently limited the cell proliferation. Treatment of Hep3B cells with 8-bromo-cAMP and SB203508 (p38 MAPK inhibitor) significantly suppressed the FACL4 expression. CONCLUSION: FACL4 is involved in the HCC tumorigenesis and both cAMP and p38 MAPK pathways are associated with the regulation of FACL4 in HCC. PMID:15849811

  15. 24-hydroxyursolic acid from the leaves of the Diospyros kaki (Persimmon) induces apoptosis by activation of AMP-activated protein kinase.

    PubMed

    Khanal, Prem; Oh, Won-Keun; Thuong, Phuong Thien; Cho, Sung Dae; Choi, Hong Seok

    2010-05-01

    There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

  16. 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

  17. Perfluorododecanoic acid-induced steroidogenic inhibition is associated with steroidogenic acute regulatory protein and reactive oxygen species in cAMP-stimulated Leydig cells.

    PubMed

    Shi, Zhimin; Feng, Yixing; Wang, Jianshe; Zhang, Hongxia; Ding, Lina; Dai, Jiayin

    2010-04-01

    Perfluorododecanoic acid (PFDoA) can be detected in environmental matrices and human serum and has been shown to inhibit testicular steroidogenesis in rats. However, the mechanisms that are responsible for the toxic effects of PFDoA remain unknown. The aims of this study were to investigate the mechanism of steroidogenesis inhibition by PFDoA and to identify the molecular target of PFDoA in Leydig cells. The effects of PFDoA on steroid synthesis in Leydig cells were assessed by radioimmunoassay. The expression of key genes and proteins in steroid biosynthesis was determined by real-time PCR and Western blot analysis. Reactive oxygen species (ROS) and hydrogen peroxide (H(2)O(2)) levels were determined using bioluminescence assays. PFDoA inhibited adenosine 3',5'-cyclophosphate (cAMP)-stimulated steroidogenesis in mouse Leydig tumor cells (mLTC-1) and primary rat Leydig cells in a dose-dependent manner. However, PFDoA (1-100 microM) did not exhibit effects on cell viability and cellular ATP levels in mLTC-1 cells. PFDoA inhibited steroidogenic acute regulatory protein (StAR) promoter activity and StAR expression at the messenger RNA (mRNA) and protein levels but did not affect mRNA levels of peripheral-type benzodiazepine receptor, cholesterol side-chain cleavage enzyme, or 3beta-hydroxysteroid dehydrogenase in cAMP-stimulated mLTC-1 cells. PFDoA treatment also resulted in increased levels of mitochondrial ROS and H(2)O(2). After excessive ROS and H(2)O(2) were eliminated in PFDoA-treated mLTC-1 cells by MnTMPyP (a superoxide dismutase analog), progesterone production was partially restored and StAR mRNA and protein levels were partially recovered. These data show that PFDoA inhibits steroidogenesis in cAMP-stimulated Leydig cells by reducing the expression of StAR through a model of action involving oxidative stress.

  18. meso-Dihydroguaiaretic acid inhibits hepatic lipid accumulation by activating AMP-activated protein kinase in human HepG2 cells.

    PubMed

    Lee, Myoung-Su; Kim, Kyung Jin; Kim, Daeyoung; Lee, Kyung-Eun; Hwang, Jae-Kwan

    2011-01-01

    Hepatic lipid accumulation is a major risk factor for dyslipidemia, nonalcoholic fatty liver disease, and insulin resistance. The present study was conducted to evaluate hypolipidemic effects of meso-dihydroguaiaretic acid (MDA), anti-oxidative and anti-inflammatory compound isolated from the Myristica fragrans HOUTT., by oil red O staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot. MDA significantly inhibited insulin-induced hepatic lipid accumulation in a dose-dependent manner. The lipid-lowering effect of MDA was accompanied by increased expression of proteins involved in fatty acid oxidation and decreased expression of lipid synthetic proteins. In addition, MDA activated AMP-activated protein kinase (AMPK) as determined by phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK. The effects of MDA on lipogenic protein expression were suppressed by pretreatment with compound C, an AMPK inhibitor. Taken together, these findings show that MDA inhibits insulin-induced lipid accumulation in human HepG2 cells by suppressing expression of lipogenic proteins through AMPK signaling, suggesting a potent lipid-lowering agent.

  19. Regulation of the laminin beta 1 (LAMB1), retinoic acid receptor beta, and bone morphogenetic protein 2 genes in mutant F9 teratocarcinoma cell lines partially deficient in cyclic AMP-dependent protein kinase activity.

    PubMed

    Shen, J; Li, C; Gudas, L J

    1997-12-01

    We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.

  20. Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid.

    PubMed Central

    Mira-Y-Lopez, R; Jaramillo, S; Jing, Y

    1998-01-01

    We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5'-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100-200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300-500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5'-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA-CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA-CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription. PMID:9560322

  1. Docosahexaenoic acid inhibits proteolytic processing of sterol regulatory element-binding protein-1c (SREBP-1c) via activation of AMP-activated kinase.

    PubMed

    Deng, Xiong; Dong, Qingming; Bridges, Dave; Raghow, Rajendra; Park, Edwards A; Elam, Marshall B

    2015-12-01

    In hyperinsulinemic states including obesity and T2DM, overproduction of fatty acid and triglyceride contributes to steatosis of the liver, hyperlipidemia and hepatic insulin resistance. This effect is mediated in part by the transcriptional regulator sterol responsive element binding protein-1c (SREBP-1c), which stimulates the expression of genes involved in hepatic fatty acid and triglyceride synthesis. SREBP-1c is up regulated by insulin both via increased transcription of nascent full-length SREBP-1c and by enhanced proteolytic processing of the endoplasmic reticulum (ER)-bound precursor to yield the transcriptionally active n-terminal form, nSREBP-1c. Polyunsaturated fatty acids of marine origin (n-3 PUFA) prevent induction of SREBP-1c by insulin thereby reducing plasma and hepatic triglycerides. Despite widespread use of n-3 PUFA supplements to reduce triglycerides in clinical practice, the exact mechanisms underlying their hypotriglyceridemic effect remain elusive. Here we demonstrate that the n-3 PUFA docosahexaenoic acid (DHA; 22:5 n-3) reduces nSREBP-1c by inhibiting regulated intramembrane proteolysis (RIP) of the nascent SREBP-1c. We further show that this effect of DHA is mediated both via activation of AMP-activated protein kinase (AMPK) and by inhibition of mechanistic target of rapamycin complex 1 (mTORC1). The inhibitory effect of AMPK on SREBP-1c processing is linked to phosphorylation of serine 365 of SREBP-1c in the rat. We have defined a novel regulatory mechanism by which n-3 PUFA inhibit induction of SREBP-1c by insulin. These findings identify AMPK as an important negative regulator of hepatic lipid synthesis and as a potential therapeutic target for hyperlipidemia in obesity and T2DM.

  2. The synthesis and characterization of environmentally-responsive water-swellable and water-soluble polymers for wastewater remediation

    NASA Astrophysics Data System (ADS)

    Armentrout, Rodney Scott

    The primary research goal is the development of new polymeric materials that demonstrate the environmentally-responsive sequestration of common water foulants, including surfactants and oils. Water-swellable and water-soluble polymers have been synthesized, structurally characterized, and their physical properties have been determined. In addition, the ability of the materials to sequester model water foulants has been evaluated. Anionic crosslinked polymer networks of 2-acrylamido-2-methyl-1-propanesulfonic acid, acrylamide, and methylene bisacrylamide have been synthesized and characterized by determining the equilibrium water contents as a function of ionic content of the polymer network. The molar ratio of bound surfactant to ionic group was determined to be less than one for all hydrogels studied, indicating an ion-exchange binding mechanism with minimal hydrophobic interactions between bound and unbound surfactant molecules is responsible for surfactant binding. Cationic crosslinked cyclopolymer networks of N,N-diallyl- N-methyl amine (DAMA) and N,N,N,N-tetraallyl ammonium chloride (TAAC) have been synthesized and characterized by determining the equilibrium water content as a function of pH. A maximum in the equilibrium water content is observed for pH-6 when the polymer is fully ionized. The solubilization of a model water foulant, p-cresol, by the polymeric surfactant, Pluronic F127, has been studied via equilibrium dialysis, dynamic light scattering and ultrafiltration experiments. It has been shown that at 25°C p-cresol is readily solubilized by F127 since the polymeric surfactant exists in a multimer conformation. Ultrafiltration experiments have demonstrated that the polymer-foulant binding interactions are largely unaffected by shear in a hollow fiber membrane. Copolymers of the zwitterionic monomer, 3-(N,N-diallyl- N-methyl ammonio) propane sulfonate (DAMAPS) and N,N-diallyl- N,N-dimethylammonium chloride (DADMAC) (the DADS series) or the p

  3. 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.

  4. Resistance to Cefepime and Cefpirome Due to a 4-Amino-Acid Deletion in the Chromosome-Encoded AmpC β-Lactamase of a Serratia marcescens Clinical Isolate

    PubMed Central

    Mammeri, Hedi; Poirel, Laurent; Bemer, Pascal; Drugeon, Henri; Nordmann, Patrice

    2004-01-01

    A multiresistant Serratia marcescens strain, HD, isolated from a patient with a urinary tract infection, was resistant to amino-, carboxy-, and ureidopenicillins, ceftazidime, and cefepime and was susceptible to cefotaxime and ceftriaxone, according to the guidelines of the NCCLS. No synergy was found between expanded-spectrum cephalosporins and clavulanic acid, according to the double-disk synergy test. The blaAmpC gene of the strain was amplified by PCR and cloned into Escherichia coli DH10B, giving rise to high-level resistance to ceftazidime, cefepime, and cefpirome. Sequencing analysis revealed that the blaAmpC gene from S. marcescens HD had a 12-nucleotide deletion compared to the blaAmpC gene from reference strain S. marcescens S3, leading to a 4-amino-acid deletion located in the H-10 helix of the β-lactamase. Kinetic analysis showed that this enzyme significantly hydrolyzed ceftazidime, cefepime, and cefpirome. This work underlined that resistance to the latest expanded-spectrum cephalosporins may be mediated by structurally modified AmpC-type β-lactamases. PMID:14982755

  5. Resistance to cefepime and cefpirome due to a 4-amino-acid deletion in the chromosome-encoded AmpC beta-lactamase of a Serratia marcescens clinical isolate.

    PubMed

    Mammeri, Hedi; Poirel, Laurent; Bemer, Pascal; Drugeon, Henri; Nordmann, Patrice

    2004-03-01

    A multiresistant Serratia marcescens strain, HD, isolated from a patient with a urinary tract infection, was resistant to amino-, carboxy-, and ureidopenicillins, ceftazidime, and cefepime and was susceptible to cefotaxime and ceftriaxone, according to the guidelines of the NCCLS. No synergy was found between expanded-spectrum cephalosporins and clavulanic acid, according to the double-disk synergy test. The bla(AmpC) gene of the strain was amplified by PCR and cloned into Escherichia coli DH10B, giving rise to high-level resistance to ceftazidime, cefepime, and cefpirome. Sequencing analysis revealed that the bla(AmpC) gene from S. marcescens HD had a 12-nucleotide deletion compared to the bla(AmpC) gene from reference strain S. marcescens S3, leading to a 4-amino-acid deletion located in the H-10 helix of the beta-lactamase. Kinetic analysis showed that this enzyme significantly hydrolyzed ceftazidime, cefepime, and cefpirome. This work underlined that resistance to the latest expanded-spectrum cephalosporins may be mediated by structurally modified AmpC-type beta-lactamases.

  6. Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

    SciTech Connect

    Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae; Hwang, Jae-Kwan

    2010-10-08

    Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepatic triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.

  7. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus*

    PubMed Central

    Bowman, Lisa; Zeden, Merve S.; Kaever, Volkhard

    2016-01-01

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5′-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. PMID:27834680

  8. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

    PubMed

    Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika

    2016-12-30

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.

  9. Effect of a high fat diet on rat adipocyte lipolysis: responses to epinephrine, forskolin, methylisobutylxanthine, dibutyryl cyclic AMP, insulin and nicotinic acid.

    PubMed

    Tepperman, H M; Dewitt, J; Tepperman, J

    1986-10-01

    An earlier report from this laboratory showed that feeding rats a high fat diet decreased epinephrine-stimulated lipolysis in their adipose tissue. Experiments were designed to explore further the effects of such diets on adipocyte response to epinephrine and to several other lipolytic and antilipolytic agents. Rats were fed diets with 67% of energy consisting of glucose or lard for 5 to 7 d. Adipocytes were prepared from epididymal fat pads and lipolysis measured by the release of glycerol into the medium during 1-h incubations. The cells from the rats fed the high fat diet showed lower lipolytic responses to stimulation by epinephrine, forskolin and dibutyryl cyclic AMP than those from rats fed the high glucose diet. The lard diet effect on the lipolytic response to isobutylmethylxanthine varied among experiments, but it also decreased it in some of them. However, the high fat diet did not induce decreased sensitivity or responsiveness to the antilipolytic effect of insulin, although previous reports have demonstrated resistance to other actions of insulin in rats fed a high fat diet. The antilipolytic effect of nicotinic acid was also similar in cells from rats fed a high fat diet to that found for cells from rats fed the high glucose diet.

  10. Short-chain fatty acids activate AMP-activated protein kinase and ameliorate ethanol-induced intestinal barrier dysfunction in Caco-2 cell monolayers.

    PubMed

    Elamin, Elhaseen E; Masclee, Ad A; Dekker, Jan; Pieters, Harm-Jan; Jonkers, Daisy M

    2013-12-01

    Short-chain fatty acids (SCFAs) have been shown to promote intestinal barrier function, but their protective effects against ethanol-induced intestinal injury and underlying mechanisms remain essentially unknown. The aim of the study was to analyze the influence of SCFAs on ethanol-induced barrier dysfunction and to examine the role of AMP-activated protein kinase (AMPK) as a possible mechanism using Caco-2 monolayers. The monolayers were treated apically with butyrate (2, 10, or 20 mmol/L), propionate (4, 20, or 40 mmol/L), or acetate (8, 40, or 80 mmol/L) for 1 h before ethanol (40 mmol/L) for 3 h. Barrier function was analyzed by measurement of transepithelial resistance and permeation of fluorescein isothiocyanate-labeled dextran. Distribution of the tight junction (TJ) proteins zona occludens-1, occludin, and filamentous-actin (F-actin) was examined by immunofluorescence. Metabolic stress was determined by measuring oxidative stress, mitochondrial function, and ATP using dichlorofluorescein diacetate, dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide, and bioluminescence assay, respectively. AMPK was knocked down by small interfering RNA (siRNA), and its activity was assessed by a cell-based ELISA. Exposure to ethanol significantly impaired barrier function compared with controls (P < 0.0001), disrupted TJ and F-actin cytoskeleton integrity, and induced metabolic stress. However, pretreatment with 2 mmol/L butyrate, 4 mmol/L propionate, and 8 mmol/L acetate significantly alleviated the ethanol-induced barrier dysfunction, TJ and F-actin disruption, and metabolic stress compared with ethanol-exposed monolayers (P < 0.0001). The promoting effects on barrier function were abolished by inhibiting AMPK using either compound C or siRNA. These observations indicate that SCFAs exhibit protective effects against ethanol-induced barrier disruption via AMPK activation, suggesting a potential for SCFAs as prophylactic and/or therapeutic factors against ethanol

  11. Sp1 Upregulates cAMP Response Element-Binding Protein Expression During Retinoic Acid-Induced Mucous Differentiation of Normal Human Bronchial Epithelial Cells

    PubMed Central

    Hong, Jeong Soo; Kim, Seung-Wook; Koo, Ja Seok

    2010-01-01

    Cyclic 3′,5′-adenosine monophosphate (cAMP) response-element (CRE) binding protein (CREB) is an important transcription factor that is differentially regulated in cells of various types. We recently reported that RA rapidly activates CREB without using retinoic acid (RA) receptors RAR and RXR in normal human tracheobronchial epithelial (NHTBE) cells. However, little is known about RA’s role in the physiologic regulation of CREB expression in the early mucous differentiation of NHTBE cells. Here, we report that RA upregulated CREB gene expression and that using 5′-serial deletion promoter analysis and mutagenesis analyses, two Sp1-binding sites located at nucleotides −217 and −150, which flank the transcription initiation site, were essential for RA induction of CREB gene transcription. Furthermore, we found that CREs located at nucleotides −119 and −98 contributed to basal promoter activity. Interestingly, RA also upregulated Sp1 in a time- and dose-dependent manner. Knockdown of endogenous Sp1 using small interfering RNA (siRNA) decreased RA-induced CREB gene expression. However, the converse was not true: knockdown of CREB using CREB siRNA did not affect RA-induced Sp1 gene expression. We conclude that RA upregulates CREB gene expression during the early stage of NHTBE cell differentiation and that RA-inducible Sp1 plays a major role in upregulating human CREB gene expression. This result implies that cooperation of these two transcription factors play a crucial role in mediating early events of normal mucous cell differentiation of bronchial epithelial cells. PMID:17937658

  12. Chicoric acid is an antioxidant molecule that stimulates AMP kinase pathway in L6 myotubes and extends lifespan in Caenorhabditis elegans.

    PubMed

    Schlernitzauer, Audrey; Oiry, Catherine; Hamad, Raphael; Galas, Simon; Cortade, Fabienne; Chabi, Béatrice; Casas, François; Pessemesse, Laurence; Fouret, Gilles; Feillet-Coudray, Christine; Cros, Gérard; Cabello, Gérard; Magous, Richard; Wrutniak-Cabello, Chantal

    2013-01-01

    Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 μM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

  13. Chicoric Acid Is an Antioxidant Molecule That Stimulates AMP Kinase Pathway in L6 Myotubes and Extends Lifespan in Caenorhabditis elegans

    PubMed Central

    Schlernitzauer, Audrey; Oiry, Catherine; Hamad, Raphael; Galas, Simon; Cortade, Fabienne; Chabi, Béatrice; Casas, François; Pessemesse, Laurence; Fouret, Gilles; Feillet-Coudray, Christine; Cros, Gérard; Cabello, Gérard; Magous, Richard; Wrutniak-Cabello, Chantal

    2013-01-01

    Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5–100 μM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases. PMID:24244361

  14. 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.

  15. 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.

  16. The vasorelaxant effect of 8(17),12E,14-labdatrien-18-oic acid involves stimulation of adenylyl cyclase and cAMP/PKA pathway: Evidences by pharmacological and molecular docking studies.

    PubMed

    Ribeiro, Luciano A A; Alencar Filho, Edilson B; Coelho, Maisa C; Silva, Bagnólia A

    2015-10-05

    The relaxant effect of 8(17),12E,14-labdatrien-18-oic acid (LBD) was investigated on isolated aortic rings and compared with forskolin (FSK), a standard and potent activator of adenylyl cyclase (AC) with relaxing effect. The presence of potassium channel blockers, such as glibenclamide (ATP-blocker), apamin (SKCa-blocker), charybdotoxin (BKCa-blocker) did not significantly affect either the LBD or FSK concentration-response curves. However, in the presence of 4-aminopyridine (KV-blocker), the relaxant effect for both diterpenes was significantly attenuated, with reduction of its relative potencies. Moreover, the relaxation induced by 8-Br-cAMP, an analog of cAMP, was also significantly attenuated in the same conditions, i.e., in the presence of 4-aminopyridine. The presence of aminophylline, a nonselective phosphodiesterase inhibitor, caused a significant increasing in the potency for both LBD and FSK. On the other hand, the presence of Rp-cAMPS, a selective PKA-inhibitor, significantly attenuated the relaxant effect of LBD. In this work, in the same experimental conditions, both labdane-type diterpenes presented remarkably similar results; FSK, however, presented a higher potency (100-fold) than LBD. Thus, the hypothesis that LBD could be a novel AC-activator emerged. To assess that hypothesis, computational molecular docking studies were performed. Crystallographic structure of adenylyl cyclase/forskolin complex (1AB8) was obtained from RSCB Protein Data Bank and used to compare the modes of interaction of the native ligand and LBD. The computational data shows many similarities between LBD and FSK concerning the interaction with the regulatory site of AC. Taken together, the results presented here pointed to LBD as a novel AC-activator.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. S-allyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway.

    PubMed

    Hwang, Yong Pil; Kim, Hyung Gyun; Choi, Jae Ho; Do, Minh Truong; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2013-08-01

    S-Allyl cysteine (SAC), a nontoxic garlic compound, has a variety of pharmacological properties, including antioxidant and hepatoprotective properties. In this report, we provide evidence that SAC prevented free fatty acid (FFA)-induced lipid accumulation and lipotoxicity in hepatocytes. SAC significantly reduced FFA-induced generation of reactive oxygen species, caspase activation and subsequent cell death. Also, SAC mitigated total cellular lipid and triglyceride accumulation in steatotic HepG2 cells. SAC significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Additionally, SAC down-regulated the levels of sterol regulatory element binding protein-1 (SREBP-1) and its target genes, including ACC and fatty acid synthase. Use of a specific inhibitor showed that SAC activated AMPK via calcium/calmodulin-dependent kinase kinase (CaMKK) and silent information regulator T1. Our results demonstrate that SAC activates AMPK through CaMKK and inhibits SREBP-1-mediated hepatic lipogenesis. Therefore, SAC has therapeutic potential for preventing nonalcoholic fatty liver disease.

  2. 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.

  3. 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.

  4. 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

  5. Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

    SciTech Connect

    Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

    2008-07-04

    AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca{sup 2+}/calmodulin-dependent protein kinase kinase (CaMKK) {beta}. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPK{alpha}1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-{kappa}B) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-{kappa}B activation.

  6. 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

  7. 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.

  8. 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.

  9. Identification of positive and negative regulatory elements involved in the retinoic acid/cAMP induction of Fgf-3 transcription in F9 cells.

    PubMed Central

    Murakami, A; Grinberg, D; Thurlow, J; Dickson, C

    1993-01-01

    The proto-oncogene Fgf-3 has been implicated as an important signalling molecule in vertebrate development. In the mouse, it is expressed for a limited time at a multitude of sites from embryonic day 7 to birth. Transcription of Fgf-3 initiates at three promoter regions resulting in the generation of various mRNAs which nevertheless all encode the same protein products. A 1.7kb DNA fragment which encompasses these regions was joined to the CAT reporter gene and shown to function as a promoter in embryonal carcinoma cells. In stable transfectants the promoter retains its retinoic acid inducibility, initiating transcription at the same cap-sites as the endogenous gene. In differentiated F9 cells, transient transfection of progressive and targeted deletion mutants of the promoter region has revealed at least two positive and three negative regulatory elements. With one exception, loss of these elements was shown to dramatically affect promoter activity in stable transfectants of F9 cells. However the promoter remained inducible by retinoic acid to differing degrees, apart from deletions encompassing PS-4A which essentially abolished promoter activity in both undifferentiated and differentiated cells. The sequences of these potential regulatory regions were further defined using DNase-I footprinting, revealing some similarities to consensus binding sites for known transcription factors. Images PMID:8265348

  10. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  11. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-06-03

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  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. 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

  16. 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.

  17. 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

  18. 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.

  19. 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].

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A.

    PubMed

    Wilderman, Andrea; Guo, Yurong; Divakaruni, Ajit S; Perkins, Guy; Zhang, Lingzhi; Murphy, Anne N; Taylor, Susan S; Insel, Paul A

    2015-09-04

    Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses, but the role of mitochondria in such responses is poorly understood. To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wild-type (WT) and kin(-) (PKA-null) murine S49 lymphoma cells. Basally, 75 proteins significantly differed in abundance between WT and kin(-) S49 cells. WT, but not kin(-), S49 cells incubated with the cAMP analog 8-(4-chlorophenylthio)adenosine cAMP (CPT-cAMP) for 16 h have (a) increased expression of mitochondria-related genes and proteins, including ones in pathways of branched-chain amino acid and fatty acid metabolism and (b) increased maximal capacity of respiration on branched-chain keto acids and fatty acids. CPT-cAMP also regulates the cellular rate of ATP-utilization, as the rates of both ATP-linked respiration and proton efflux are decreased in WT but not kin(-) cells. CPT-cAMP protected WT S49 cells from glucose or glutamine deprivation, In contrast, CPT-cAMP did not protect kin(-) cells or WT cells treated with the PKA inhibitor H89 from glutamine deprivation. Under basal conditions, the mitochondrial structure of WT and kin(-) S49 cells is similar. Treatment with CPT-cAMP produced apoptotic changes (i.e. decreased mitochondrial density and size and loss of cristae) in WT, but not kin(-) cells. Together, these findings show that cAMP acts via PKA to regulate multiple aspects of mitochondrial function and structure. Mitochondrial perturbation thus likely contributes to cAMP/PKA-mediated cellular responses.

  6. "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.

  7. “Store-operated” cAMP signaling contributes to Ca2+-activated Cl− secretion in T84 colonic cells

    PubMed Central

    Nichols, Jonathan M.; Maiellaro, Isabella; Abi-Jaoude, Joanne; Curci, Silvana

    2015-01-01

    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 Ca2+-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca2+, and the existence of apical Ca2+-dependent Cl− channels in the native colonic epithelium is controversial, leaving the identity of the Ca2+-activated component unresolved. We recently showed that decreasing free Ca2+ concentration ([Ca2+]) 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 Ca2+ sensor STIM1 and does not depend on changes in cytosolic Ca2+. Here we assessed the degree to which SOcAMPS participates in Ca2+-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 Ca2+-free conditions, the Ca2+-releasing agonist carbachol and Ca2+ ionophore increased Isc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca2+ 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 Ba2+ or flufenamic acid. We propose that a discrete component of the “Ca2+-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 Ca2+ homeostasis, leading to diarrhea. PMID:26316590

  8. 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.

  9. Involvement of the Pepper Antimicrobial Protein CaAMP1 Gene in Broad Spectrum Disease Resistance1[C][OA

    PubMed Central

    Lee, Sung Chul; Hwang, In Sun; Choi, Hyong Woo; Hwang, Byung Kook

    2008-01-01

    Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (Allium cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. campestris pv vesicatoria and Colletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thaliana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad-spectrum resistance to bacterial and fungal pathogen infection. PMID:18676663

  10. 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

  11. Enterobacter cloacae with a novel variant of ACT AmpC beta-lactamase originating from glaucous gull (Larus hyperboreus) in Svalbard.

    PubMed

    Literak, Ivan; Manga, Ivan; Wojczulanis-Jakubas, Katarzyna; Chroma, Magdalena; Jamborova, Ivana; Dobiasova, Hana; Sedlakova, Miroslava Htoutou; Cizek, Alois

    2014-07-16

    We aimed at Escherichia coli and Enterobacter cloacae isolates resistant to cephalosporins and fluoroquinolones and Salmonella isolates in wild birds in Arctic Svalbard, Norway. Cloacal swabs of little auks (Alle alle, n=215) and samples of faeces of glaucous gulls (Larus hyperboreus, n=15) were examined. Inducible production of AmpC enzyme was detected in E. cloacae KW218 isolate. Sequence analysis of the 1146 bp PCR product of the ampC gene from this isolate revealed 99% sequence homology with the blaACT-14 and blaACT-5 AmpC beta-lactamase genes. Four, respectively six of the identified single nucleotide polymorphisms generated amino acid substitutions in the amino acid chain. As the ampC sequence polymorphism in the investigated E. cloacae strain was identified as unique, we revealed a novel variant of the ampC beta-lactamase gene blaACT-23.

  12. 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.

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

    PubMed

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

    2013-01-01

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

  14. A comparative study of the neutral and acidic polysaccharides from Allium macrostemon Bunge.

    PubMed

    Zhang, Zhanjun; Wang, Fuhua; Wang, Mingchun; Ma, Liping; Ye, Hong; Zeng, Xiaoxiong

    2015-03-06

    Neutral and acidic polysaccharides, named AMP40N and AMP40S respectively, were isolated and purified from the dried bulbs of Allium macrostemon Bunge. Both of them showed a single and symmetrically sharp peak, indicating they were homogeneous polysaccharides. Molecular weights of AMP40N and AMP40S were determined to be 18.2 and 105.1 kDa, respectively. AMP40N was composed of arabinose and glucose, while AMP40S was composed of rhamnose, arabinose, glucose and galactose and a certain amount of uronic acid. FT-IR, periodic acid oxidation, Smith degradation, methylation and GC-MS analysis revealed that non-reducing terminal and →2,6)-Glc-(1→ existed in AMP40N and AMP40S. The glycosidic linkage of arabinose in AMP40N was →2)-Ara-(1→, whereas it was Ara-(1→ in AMP40S. AMP40S had (1→2)-linked l-rhamnose residue. Both AMP40N and AMP40S exhibited strong anti-tumor potential against human gastric carcinoma cells BGC-823, in particular, AMP40S presented significantly higher inhibitory rate of 85.94% than AMP40N of 52.63%.

  15. 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.

  16. 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.

  17. 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.

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  1. Astrocyte arachidonate and palmitate uptake and metabolism is differentially modulated by dibutyryl-cAMP treatment.

    PubMed

    Seeger, D R; Murphy, C C; Murphy, E J

    2016-07-01

    Astrocytes play a vital role in brain lipid metabolism; however the impact of the phenotypic shift in astrocytes to a reactive state on arachidonic acid metabolism is unknown. Therefore, we determined the impact of dibutyryl-cAMP (dBcAMP) treatment on radiolabeled arachidonic acid ([1-(14)C]20:4n-6) and palmitic acid ([1-(14)C]16:0) uptake and metabolism in primary cultured murine cortical astrocytes. In dBcAMP treated astrocytes, total [1-(14)C]20:4n-6 uptake was increased 1.9-fold compared to control, while total [1-(14)C]16:0 uptake was unaffected. Gene expression of long-chain acyl-CoA synthetases (Acsl), acyl-CoA hydrolase (Acot7), fatty acid binding protein(s) (Fabp) and alpha-synuclein (Snca) were determined using qRT-PCR. dBcAMP treatment increased expression of Acsl3 (4.8-fold) and Acsl4 (1.3-fold), which preferentially use [1-(14)C]20:4n-6 and are highly expressed in astrocytes, consistent with the increase in [1-(14)C]20:4n-6 uptake. However, expression of Fabp5 and Fabp7 were significantly reduced by 25% and 45%, respectively. Acot7 (20%) was also reduced, suggesting dBcAMP treatment favors acyl-CoA formation. dBcAMP treatment enhanced [1-(14)C]20:4n-6 (2.2-fold) and [1-(14)C]16:0 (1.6-fold) esterification into total phospholipids, but the greater esterification of [1-(14)C]20:4n-6 is consistent with the observed uptake through increased Acsl, but not Fabp expression. Although total [1-(14)C]16:0 uptake was not affected, there was a dramatic decrease in [1-(14)C]16:0 in the free fatty acid pool as esterification into the phospholipid pool was increased, which is consistent with the increase in Acsl3 and Acsl4 expression. In summary, our data demonstrates that dBcAMP treatment increases [1-(14)C]20:4n-6 uptake in astrocytes and this increase appears to be due to increased expression of Acsl3 and Acsl4 coupled with a reduction in Acot7 expression.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Effect of ovine luteinizing hormone (oLH) charge isoforms on VEGF and cAMP production.

    PubMed

    Montero-Pardo, Arnulfo; Diaz, Daniel; Olivares, Aleida; González-Padilla, Everardo; Murcia, Clara; Gómez-Chavarín, Margarita; Gutiérrez-Ospina, Gabriel; Perera-Marín, Gerardo

    2015-12-01

    Although an increase in VEGF expression and synthesis in association with LH has been established; it is unknown if all LH isoforms act similarly. This study evaluated the production of cAMP and VEGF among LH isoforms in two in vitro bioassays. The LH was obtained from hypophyses and the group of isoforms was isolated by chromatofocusing. cAMP production was assessed using the in vitro bioassay of HEK-293 cells and VEGF production was evaluated in granulosa cells. Immunological activity was measured with a homologous RIA. Immunoactivity and bioactivity for each isoform were compared against a standard, by estimating the IC50 and the EC50. The basic isoforms were more immunoactive than the standard. The neutral and the moderately acidic had an immunological activity similar to the standard. The acidic isoform was the least immunoreactive. cAMP production at the EC50 dose was similar among the basic isoforms, the moderately acidic and the standard; for the neutral and the acidic, the EC50 dose was higher. It was observed that compared with the control, VEGF production at the lowest LH dose was no different in the standard and each isoform. In the intermediate dose, a positive response was caused in the standard and the neutral and basic isoforms. Although the acidic isoform showed a dose-dependent response, it was not significant relative to the control. In conclusion, the basic isoform generated the greatest cAMP and VEGF production, similar to the reference standard, and the acidic the smallest.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. [Detection of plasmid-mediated AmpC beta-lactamase production in Escherichia coli and Klebsiella spp. isolates].

    PubMed

    Balıkçı, Hilal; Açıkgöz, Z Cibali; Güvenman, Selda; Celikbilek, Nevreste; Ozdem, Birsen

    2014-01-01

    This study was conducted to obtain data about prevalence of plasmid-mediated AmpC (pAmpC), efficacy of various phenotypic tests applied for the detection of pAmpC-mediated resistance and the enzyme types responsible for this resistance. A total of 1316 isolates (1030 Escherichia coli and 286 Klebsiella spp.) obtained from the clinical samples sent to our laboratory between 2008-2011 period, from various inpatient and outpatient clinics and intensive care units, were included in this study. Antimicrobial susceptibility profiles of the isolates were determined by using Kirby-Bauer disk diffusion method. Production of pAmpC was phenotypically investigated by Boronic Acid Inhibition Test (BAIT), Cefoxitin Hodge Test (CHT) and AmpC Disk Test (ACDT) in a total of 36 (2.7%) cefoxitin-resistant isolates (77% were E.coli, 23% were K.pneumoniae); and by synergy tests with or without AmpC inhibitors in a total of 165 (88% were E.coli, 12% were K.pneumoniae) cefoxitin-susceptible, third generation cephalosporin-resistant (S3R) isolates. For the detection of pAmpC genes a multiplex-PCR protocol was applied to the isolates found positive at least by one of the phenotypic tests. CHT, ACDT and BAIT were found positive in 21 (58%), 18 (50%) and 7 (19%) of the 36 cefoxitin-resistant isolates, respectively. In only 10 (27.7%) of these isolates (all were E.coli strains), pAmpC presence was detected by PCR; and the enzyme produced was assessed as CMY-2. Based on the positive PCR results; specificity rates of the phenotypic tests, BAIT, ACDT and CHT were 97%, 69% and 58%; while the sensitivity rates were 50%, 100% and 100%, respectively. Our data indicated that, pAmpC prevalence (10/1316, 0.8%) detected in this study, did not seem to be an important problem yet, in the population screened. However, since the first detection of this resistance was in 2010, it should be considered as a sign to get necessary precautions preventing its spread. In conclusion, none of the phenotypic methods

  14. 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.

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

    PubMed

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

    2010-11-01

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

  16. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  17. Adenyl cyclases and cAMP in plant signaling - past and present.

    PubMed

    Gehring, Chris

    2010-06-25

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

  18. The Effect of Cyclic AMP on the Permeability Characteristics of the Escherichia coli Membrane System

    DTIC Science & Technology

    1977-09-21

    Dobigosz. 1974. Stimulation of cytochrome syndes.is i•• 7sche.ichia coli by cyclic AMP. Arch. Bioche -.. B3iophlw. L62:5S5-601. 2. Ezzell, J. W., and W. J...and fatty acid composition in Escherichia col. by cyclic .M4P roce’ pj-oteln. Arce. Bioch •m. Biophys. 175:295-302. 6. bills, S. S., and W. J. Duobr

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

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

    PubMed

    Alasbahi, R H; Melzig, M F

    2012-01-01

    protoberberine alkaloid palmatine on the active ion transport across rat colonic epithelium, the inhibitory effect of retinoic acid on HIV-1-induced podocyte proliferation, the whitening activity of luteolin, the effect of cilostazol on nitric oxide production, an effect that is involved in capillary-like tube formation in human aortic endothelial cells, the apoptotic effect of bullatacin, the effects of paraoxon and chlorpyrifos oxon on nervous system. Moreover, cAMP was found to play a role in acute and chronic exposure to ethanol, in morphine dependence and withdrawal and in behavioral sensitization to cocaine as well as in the protection against cisplatin-induced oxidative injuries.

  5. Global Role of Cyclic AMP Signaling in pH-Dependent Responses in Candida albicans

    PubMed Central

    Hollomon, Jeffrey M.; Grahl, Nora; Willger, Sven D.; Koeppen, Katja

    2016-01-01

    ABSTRACT Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output. Active alleles of ras1 do not suppress the hyphal growth defect at low pH, while dibutyryl cAMP partially rescues filamentous growth at low pH in a cyr1 mutant. These observations are consistent with Ras1-independent downregulation of Cyr1 by low pH. We also report that extracellular pH leads to rapid and prolonged decreases in intracellular pH, and these changes may contribute to reduced cAMP signaling by reducing intracellular bicarbonate pools. Transcriptomics analyses found that the loss of Cyr1 at either acidic or neutral pH leads to increases in transcripts involved in carbohydrate catabolism and protein translation and glycosylation and decreases in transcripts involved in oxidative metabolism, fluconazole transport, metal transport, and biofilm formation. Other pathways were modulated in pH-dependent ways. Our findings indicate that cAMP has a global role in pH-dependent responses, and this effect is mediated, at least in part, through Cyr1 in a Ras1-independent fashion. IMPORTANCE Candida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an

  6. Global Role of Cyclic AMP Signaling in pH-Dependent Responses in Candida albicans.

    PubMed

    Hollomon, Jeffrey M; Grahl, Nora; Willger, Sven D; Koeppen, Katja; Hogan, Deborah A

    2016-01-01

    Candida albicans behaviors are affected by pH, an important environmental variable. Filamentous growth is a pH-responsive behavior, where alkaline conditions favor hyphal growth and acid conditions favor growth as yeast. We employed filamentous growth as a tool to study the impact of pH on the hyphal growth regulator Cyr1, and we report that downregulation of cyclic AMP (cAMP) signaling by acidic pH contributes to the inhibition of hyphal growth in minimal medium with GlcNAc. Ras1 and Cyr1 are generally required for efficient hyphal growth, and the effects of low pH on Ras1 proteolysis and GTP binding are consistent with diminished cAMP output. Active alleles of ras1 do not suppress the hyphal growth defect at low pH, while dibutyryl cAMP partially rescues filamentous growth at low pH in a cyr1 mutant. These observations are consistent with Ras1-independent downregulation of Cyr1 by low pH. We also report that extracellular pH leads to rapid and prolonged decreases in intracellular pH, and these changes may contribute to reduced cAMP signaling by reducing intracellular bicarbonate pools. Transcriptomics analyses found that the loss of Cyr1 at either acidic or neutral pH leads to increases in transcripts involved in carbohydrate catabolism and protein translation and glycosylation and decreases in transcripts involved in oxidative metabolism, fluconazole transport, metal transport, and biofilm formation. Other pathways were modulated in pH-dependent ways. Our findings indicate that cAMP has a global role in pH-dependent responses, and this effect is mediated, at least in part, through Cyr1 in a Ras1-independent fashion. IMPORTANCECandida albicans is a human commensal and the causative agent of candidiasis, a potentially invasive and life-threatening infection. C. albicans experiences wide changes in pH during both benign commensalism (a common condition) and pathogenesis, and its morphology changes in response to this stimulus. Neutral pH is considered an activator

  7. Epidemiology of extended-spectrum β-lactamase, AmpC, and carbapenemase production in Proteus mirabilis.

    PubMed

    Datta, Priya; Gupta, Varsha; Arora, Shilpa; Garg, Shivani; Chander, Jagdish

    2014-01-01

    Proteus mirabilis strains that produce extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase pose potential threats to patient care because most clinical diagnostic laboratories may not attempt to detect these three major groups of enzymes. Therefore, the objective of this study was to ascertain if P. mirabilis isolates collected from our heathcare facility possess various mechanisms of resistance to β-lactams (i.e., ESBL, AmpC, and carbapenemases) and to additionally arrive at conclusions regarding concurrent testing for these three mechanism of drug resistance in order to reduce cost and time in routine diagnostic testing. Between January 2011 and June 2011, 60 consecutive non-repeated strains of P. mirabilis were evaluated for production of ESBLs, AmpC β-lactamases, and carbapenemases. Of these, 36 isolates were found to be ESBL producers, and 7 (12%) were positive for production of AmpC β-lactamases and ESBLs. Therefore, 19.4% of ESBL-producing Proteus strains coproduced AmpC enzymes. The modified Hodge test confirmed carbapenemase production in only 1 isolate (1.7%), which was also ESBL- and AmpC-positive. The clinical impact of additional AmpC expression in ESBL-producing P. mirabilis results in a newly acquired resistance to β-lactamase inhibitors. In addition, to save time and costs, we recommend the use of cefepime/cefepime-clavulanate or boronic acid for the ESBL detection but in only those strains that were positive for ESBL by screening and negative by confirmatory tests.

  8. Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes.

    PubMed

    Zhou, Libin; Wang, Xiao; Yang, Ying; Wu, Ling; Li, Fengying; Zhang, Rong; Yuan, Guoyue; Wang, Ning; Chen, Mingdao; Ning, Guang

    2011-04-01

    Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of (3)[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

  9. Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

    PubMed Central

    Berrazeg, M.; Jeannot, K.; Ntsogo Enguéné, Véronique Yvette; Broutin, I.; Loeffert, S.; Fournier, D.

    2015-01-01

    Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting. PMID:26248364

  10. 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.

  11. 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

  12. Antimicrobial peptide (Cn-AMP2) from liquid endosperm of Cocos nucifera forms amyloid-like fibrillar structure.

    PubMed

    Gour, Shalini; Kaushik, Vibha; Kumar, Vijay; Bhat, Priyanka; Yadav, Subhash C; Yadav, Jay K

    2016-04-01

    Cn-AMP2 is an antimicrobial peptide derived from liquid endosperm of coconut (Cocos nucifera). It consists of 11 amino acid residues and predicted to have high propensity for β-sheet formation that disposes this peptide to be amyloidogenic. In the present study, we have examined the amyloidogenic propensities of Cn-AMP2 in silico and then tested the predictions under in vitro conditions. The in silico study revealed that the peptide possesses high amyloidogenic propensity comparable with Aβ. Upon solubilisation and agitation in aqueous buffer, Cn-AMP2 forms visible aggregates that display bathochromic shift in the Congo red absorbance spectra, strong increase in thioflavin T fluorescence and fibrillar morphology under transmission electron microscopy. All these properties are typical of an amyloid fibril derived from various proteins/peptides including Aβ.

  13. [Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism].

    PubMed

    Makuch, Edyta; Matuszyk, Janusz

    2012-07-20

    PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

  14. An investigation of fluidized bed electrowinning of cobalt using 50 and 1000 Amp cells

    NASA Astrophysics Data System (ADS)

    Dubrovsky, M.; Evans, J. W.

    1991-12-01

    50 Amp and 1000 Amp cells equipped with fluidized bed cathodes were used to investigate the electrowinning of cobalt from sulfate solutions. The catholytes employed ranged in cobalt concentration from 100 to 4.8 grams per liter of cobalt and from acid (pH ≏1) to near neutral (pH-6). Superficial current densities up to 1.09 A cm-2 were used. The cells were equipped with a nearly impermeable diaphragm, permitting the use of an anolyte of composition different from that of the catholyte. The current efficiency for cobalt deposition (as conveniently determined by measuring the rate of hydrogen evolution), electrical energy consumption, and appearance of the deposit were studied as a function of catholyte composition. Reasonable current efficiencies were observed. The electrical energy consumptions were much higher than that of conventional electrowinning, but this was shown to be due to the anode chamber and diaphragm resistance losses rather than the fluidized cathode.

  15. An investigation of fluidized bed electrowinning of cobalt using 50 and 1000 Amp cells

    NASA Astrophysics Data System (ADS)

    Dubrovsky, M.; Evans, J. W.

    1982-09-01

    50 Amp and 1000 Amp cells equipped with fluidized bed cathodes were used to investigate the electrowinning of cobalt from sulfate solutions. The catholytes employed ranged in cobalt concentration from 100 to 4.8 grams per liter of cobalt and from acid (pH ≃1) to near neutral (pH ≃6). Superficial current densities up to 1.09 A cm-2 were used. The cells were equipped with a nearly impermeable diaphragm, permitting the use of an anolyte of composition different from that of the catholyte. The current efficiency for cobalt deposition (as conveniently determined by measuring the rate of hydrogen evolution), electrical energy consumption, and appearance of the deposit were studied as a function of catholyte composition. Reasonable current efficiencies were observed. The electrical energy consumptions were much higher than that of conventional electrowinning, but this was shown to be due to the anode chamber and diaphragm resistance losses rather than the fluidized cathode.

  16. Export of cyclic AMP by avian red cells and inhibition by prostaglandin A/sub 1/

    SciTech Connect

    Heasley, L.E.

    1985-01-01

    The mechanism by which PGA/sub 1/ inhibits cAMP export by avian red cells was studied, to provide details on the molecular mechanism of a prostaglandin action and on the process of cAMP export itself. The interaction of PGA/sub 1/ with pigeon red cells is a multi-step process of uptake, metabolism and secretion. (/sup 3/H)PGA rapidly enters red cells and is promptly metabolized (V/sub max/ greater than or equal to 1 nmol/min/10/sup 7/ cells) to a compound (5) that remains in the aqueous layer after ethyl acetate extraction. Chromatographic analyses, amino acid content and fast atom bombardment mass spectrometry reveal that the polar metabolite is conjugated with glutathione (PGA/sub 1/-GSH) at C-11 via a thioether bond and is largely (80%) reduced to the C-9 hydroxyl derivative.

  17. 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.

  18. 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

  19. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  1. 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.

  2. 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.

  3. 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

  4. Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes.

    PubMed Central

    Ito, Y; Uchijima, Y; Ariga, M; Seki, T; Takenaka, A; Hakuno, F; Takahashi, S I; Ariga, T; Noguchi, T

    1997-01-01

    The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175-180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90-95 kDa proteins, including the insulin receptor beta-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor beta-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Sensing of energy and nutrients by AMP-activated protein kinase.

    PubMed

    Hardie, D Grahame

    2011-04-01

    AMP-activated protein kinase (AMPK) is a cellular energy sensor that exists in almost all eukaryotes. Genetic studies in lower eukaryotes suggest that the ancestral role of AMPK was in response to starvation for a carbon source and that AMPK is involved in life-span extension in response to caloric restriction. In mammals, AMPK is activated by an increasing cellular AMP:ATP ratio (which signifies a decrease in energy) caused by metabolic stresses that interfere with ATP production (eg, hypoxia) or that accelerate ATP consumption (eg, muscle contraction). Because glucose deprivation can increase the AMP:ATP ratio, AMPK can also act as a glucose sensor. AMPK activation occurs by a dual mechanism that involves allosteric activation and phosphorylation by upstream kinases. Once activated, AMPK switches on catabolic pathways that generate ATP (eg, the uptake and oxidation of glucose and fatty acids and mitochondrial biogenesis) while switching off ATP-consuming, anabolic pathways (eg, the synthesis of lipids, glucose, glycogen, and proteins). In addition to the acute effects via direct phosphorylation of metabolic enzymes, AMPK has longer-term effects by regulating transcription. These features make AMPK an ideal drug target in the treatment of metabolic disorders such as insulin resistance and type 2 diabetes. The antidiabetic drug metformin (which is derived from an herbal remedy) works in part by activating AMPK, whereas many xenobiotics or "nutraceuticals," including resveratrol, quercetin, and berberine, are also AMPK activators. Most of these agents activate AMPK because they inhibit mitochondrial function.

  10. Phenolic diterpenes from rosemary suppress cAMP responsiveness of gluconeogenic gene promoters.

    PubMed

    Yun, Young Sook; Noda, Sachie; Shigemori, Genta; Kuriyama, Ryunosuke; Takahashi, Shigeru; Umemura, Mariko; Takahashi, Yuji; Inoue, Hideshi

    2013-06-01

    The cAMP/protein kinase A/cAMP response element (CRE)-binding protein pathway is important for various physiological aspects including regulation of gluconeogenic gene expression. Rosemary, a well-known herb, has been reported to decrease blood glucose levels. We found that methanol extracts of rosemary suppressed forskolin (FSK)-stimulated luciferase expression under the control of CRE, as well as the promoters for cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and glucose-6-phosphatase (G6Pase) catalytic subunit genes in human hepatoma HepG2 cells. Three abietane-type diterpenes and two flavonoids were isolated from the rosemary extracts. Among these, 7-O-methylrosmanol (1) and royleanonic acid (3) effectively suppressed FSK-induced luciferase expression under the control of the CRE, PEPCK-C and G6Pase gene promoters. PEPCK-C and G6Pase, which play a key role in the homeostatic regulation of blood glucose levels, are important for managing type II diabetes mellitus. Therefore, the ability of rosemary and its components to suppress cAMP responsiveness of the PEPCK-C or G6Pase gene may contribute to its antihyperglycemic activity.

  11. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

    PubMed

    Couve, A; Thomas, P; Calver, A R; Hirst, W D; Pangalos, M N; Walsh, F S; Smart, T G; Moss, S J

    2002-05-01

    GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

  12. Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

    PubMed

    Takahashi, Toshimasa; Shibasaki, Tadao; Takahashi, Harumi; Sugawara, Kenji; Ono, Aika; Inoue, Naoko; Furuya, Toshio; Seino, Susumu

    2013-10-22

    Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

  13. 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

  14. 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

  15. Signaling transcript profile of the asexual intraerythrocytic development cycle of Plasmodium falciparum induced by melatonin and cAMP

    PubMed Central

    Rozanski, Andrei; Parreira, Kleber S.; Moraes, Miriam S.; Martins, David C.; Hashimoto, Ronaldo F.; Galante, Pedro A.F.; Garcia, Célia R.S.

    2016-01-01

    According to the World Health Organization (WHO), Plasmodium falciparum is the deadliest parasite among all species. This parasite possesses the ability to sense molecules, including melatonin (MEL) and cAMP, and modulate its cell cycle accordingly. MEL synchronizes the development of this malaria parasite by activating several cascades, including the generation of the second messenger cAMP. Therefore, we performed RNA sequencing (RNA-Seq) analysis in P. falciparum erythrocytic stages (ring, trophozoite and schizont) treated with MEL and cAMP. To investigate the expression profile of P. falciparum genes regulated by MEL and cAMP, we performed RNA-Seq analysis in three P. falciparum strains (control, 3D7; protein kinase 7 knockout, PfPK7-; and PfPK7 complement, PfPK7C). In the 3D7 strain, 38 genes were differentially expressed upon MEL treatment; however, none of the genes in the trophozoite (T) stage PfPK7- knockout parasites were differentially expressed upon MEL treatment for 5 hours compared to untreated controls, suggesting that PfPK7 may be involved in the signaling leading to differential gene expression. Moreover, we found that MEL modified the mRNA expression of genes encoding membrane proteins, zinc ion-binding proteins and nucleic acid-binding proteins, which might influence numerous functions in the parasite. The RNA-Seq data following treatment with cAMP show that this molecule modulates different genes throughout the intraerythrocytic cycle, namely, 75, 101 and 141 genes, respectively, in the ring (R), T and schizont (S) stages. Our results highlight P. falciparum's perception of the external milieu through the signaling molecules MEL and cAMP, which are able to drive to changes in gene expression in the parasite. PMID:28050233

  16. 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.

  17. Phenotypic and molecular characterization of plasmid mediated AmpC β-lactamases among Escherichia coli, Klebsiella spp., and Proteus mirabilis isolated from urinary tract infections in Egyptian hospitals.

    PubMed

    Helmy, Mai M; Wasfi, Reham

    2014-01-01

    The incidence of resistance by Enterobacteriaceae to β-lactam/β-lactamase inhibitors combination is increasing in Egypt. Three phenotypic techniques, comprising AmpC disk diffusion and inhibition dependent methods using phenylboronic acid (PBA) and cloxacillin, were compared to PCR based method for detection of plasmid mediated AmpC β-lactamase in common urinary tract isolates. A total of 143 isolates, including E. coli, Klebsiella pneumonia, and Proteus mirabilis, were collected from urinary tract infections cases in Egyptian hospitals. Plasmid encoded AmpC genes were detected by PCR in 88.46% of cefoxitin resistant isolates. The most prevalent AmpC gene family was CIT including CMY-2, CMY-4, and two CMY-2 variants. The second prevalent gene was DHA-1 which was detected in E. coli and Klebsiella pneumonia. The genes EBC, FOX, and MOX were also detected but in small percentage. Some isolates were identified as having more than one pAmpC gene. The overall sensitivity and specificity of phenotypic tests for detection of AmpC β-lactamase showed that AmpC disk diffusion and inhibition dependent method by cloxacillin were the most sensitive and the most specific disk tests. PCR remains the gold standard for detection of AmpC β-lactamases. This study represents the first report of CMY-2 variants of CMY-42 and CMY-102 β-lactamase-producing E. coli, Klebsiella pneumonia, and Proteus mirabilis isolates in Egypt.

  18. Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor.

    PubMed

    Süsstrunk, U; Pidoux, J; Taubert, S; Ullmann, A; Thompson, C J

    1998-10-01

    In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3',5' monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 microM) found in the medium of MT1110 cultures, suggested that it may serve as a

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. Structure-based Design and In-Parallel Synthesis of Inhibitors of AmpC b-lactamase

    SciTech Connect

    Tondi, D.; Powers, R.A.; Negri, M.C.; Caselli, M.C.; Blazquez, J.; Costi, M.P.; Shoichet, B.K.

    2010-03-08

    Group I {beta}-lactamases are a major cause of antibiotic resistance to {beta}-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic {beta}-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I {beta}-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of {beta}-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K{sub i} 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K{sub i} values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 {angstrom} resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from {beta}-lactams but

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli.

    PubMed

    Hufnagel, David A; Evans, Margery L; Greene, Sarah E; Pinkner, Jerome S; Hultgren, Scott J; Chapman, Matthew R

    2016-12-15

    The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP

  10. Induction of a Torpor-Like State by 5’-AMP Does Not Depend on H2S Production

    PubMed Central

    Dugbartey, George J.; Bouma, Hjalmar R.; Strijkstra, Arjen M.; Boerema, Ate S.; Henning, Robert H.

    2015-01-01

    Background Therapeutic hypothermia is used to reduce ischemia/reperfusion injury (IRI) during organ transplantation and major surgery, but does not fully prevent organ injury. Interestingly, hibernating animals undergo repetitive periods of low body temperature called ‘torpor’ without signs of organ injury. Recently, we identified an essential role of hydrogen sulfide (H2S) in entrance into torpor and preservation of kidney integrity during hibernation. A torpor-like state can be induced pharmacologically by injecting 5’-Adenosine monophosphate (5’-AMP). The mechanism by which 5’-AMP leads to the induction of a torpor-like state, and the role of H2S herein, remains to be unraveled. Therefore, we investigated whether induction of a torpor-like state by 5-AMP depends on H2S production. Methods To study the role of H2S on the induction of torpor, amino-oxyacetic acid (AOAA), a non-specific inhibitor of H2S, was administered before injection with 5'-AMP to block endogenous H2S production in Syrian hamster. To assess the role of H2S on maintenance of torpor induced by 5’-AMP, additional animals were injected with AOAA during torpor. Key Results During the torpor-like state induced by 5’-AMP, the expression of H2S- synthesizing enzymes in the kidneys and plasma levels of H2S were increased. Blockade of these enzymes inhibited the rise in the plasma level of H2S, but neither precluded torpor nor induced arousal. Remarkably, blockade of endogenous H2S production was associated with increased renal injury. Conclusions Induction of a torpor-like state by 5’-AMP does not depend on H2S, although production of H2S seems to attenuate renal injury. Unraveling the mechanisms by which 5’-AMP reduces the metabolism without organ injury may allow optimization of current strategies to limit (hypothermic) IRI and improve outcome following organ transplantation, major cardiac and brain surgery. PMID:26295351

  11. Evolutionary Adaptation of the Essential tRNA Methyltransferase TrmD to the Signaling Molecule 3′,5′-cAMP in Bacteria*

    PubMed Central

    Agrebi, Rym; Bellows, Lauren E.; Collet, Jean-François; Kaever, Volkhard

    2017-01-01

    The nucleotide signaling molecule 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3′,5′-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3′,5′-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life as a tight 3′,5′-cAMP-binding protein. TrmD enzymes are known to use S-adenosyl-l-methionine (AdoMet) as substrate; we have shown that 3′,5′-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2′,3′-cAMP but not 3′,5′-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3′,5′-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis, and biochemical analyses revealed that the highly conserved Tyr-86 residue in E. coli TrmD is essential to discriminate between 3′,5′-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3′,5′-cAMP. Altogether this further indicates that S. aureus does not produce 3′,5′-cAMP, which would otherwise competitively inhibit an essential enzyme. PMID:27881678

  12. Functional analyses of AmpC beta-lactamase through differential stability.

    PubMed Central

    Beadle, B. M.; McGovern, S. L.; Patera, A.; Shoichet, B. K.

    1999-01-01

    Despite decades of intense study, the complementarity of beta-lactams for beta-lactamases and penicillin binding proteins is poorly understood. For most of these enzymes, beta-lactam binding involves rapid formation of a covalent intermediate. This makes measuring the equilibrium between bound and free beta-lactam difficult, effectively precluding measurement of the interaction energy between the ligand and the enzyme. Here, we explore the energetic complementarity of beta-lactams for the beta-lactamase AmpC through reversible denaturation of adducts of the enzyme with beta-lactams. AmpC from Escherichia coli was reversibly denatured by temperature in a two-state manner with a temperature of melting (Tm) of 54.6 degrees C and a van't Hoff enthalpy of unfolding (deltaH(VH)) of 182 kcal/mol. Solvent denaturation gave a Gibbs free energy of unfolding in the absence of denaturant (deltaG(u)H2O) of 14.0 kcal/mol. Ligand binding perturbed the stability of the enzyme. The penicillin cloxacillin stabilized AmpC by 3.2 kcal/mol (deltaTm = +5.8 degrees C); the monobactam aztreonam stabilized the enzyme by 2.7 kcal/mol (deltaTm = +4.9 degrees C). Both acylating inhibitors complement the active site. Surprisingly, the oxacephem moxalactam and the carbapenem imipenem both destabilized AmpC, by 1.8 kcal/mol (deltaTm = -3.2 degrees C) and 0.7 kcal/mol (deltaTm = -1.2 degrees C), respectively. These beta-lactams, which share nonhydrogen substituents in the 6(7)alpha position of the beta-lactam ring, make unfavorable noncovalent interactions with the enzyme. Complexes of AmpC with transition state analog inhibitors were also reversibly denatured; both benzo(b)thiophene-2-boronic acid (BZBTH2B) and p-nitrophenyl phenylphosphonate (PNPP) stabilized AmpC. Finally, a catalytically inactive mutant of AmpC, Y150F, was reversibly denatured. It was 0.7 kcal/mol (deltaTm = -1.3 degrees C) less stable than wild-type (WT) by thermal denaturation. Both the cloxacillin and the moxalactam

  13. CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

    PubMed

    Maronde, E; Wicht, H; Taskén, K; Genieser, H G; Dehghani, F; Olcese, J; Korf, H W

    1999-10-01

    Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

  14. Influence of cyclic nucleotides (cAMP) on inositol phospholipid (InsPL) metabolism in cultured mesangial (MS) cells

    SciTech Connect

    Troyer, D.A.; Venkatachalam, M.A.; Bonventre, J.V.; Kreisberg, J.I.

    1986-03-01

    Elevation of cAMP inhibits hormone-induced contraction of MS cells, and in other cell types, reduces stimulated InsPL metabolism. The authors found that neither isobutylmethylxanthine (MIX, 0.5 mM), which increases MS cell cAMP levels 4-fold, nor forskolin (100 ..mu..M) altered vasopressin (VP, 10 nM) induced release of /sup 3/H-inositol trisphosphate from prelabelled MS cells. Also, maneuvers which elevated cAMP did not block the VP-induced rise of intracellular calcium as measured by quin-2. Further, neither MIX nor isoproterenol affected the stimulation of glycolysis by VP as measured by lactic acid production. MIX diminished VP stimulated /sup 32/P orthophosphate (/sup 32/P) incorporation into phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol. The /sup 32/P content in phosphoinositides of cells treated with MIX and VP was 65% of that in cells treated with VP only. However, the authors found that the specific activity of /sup 32/P in ATP in the presence of MIX + VP was 74% of that with VP alone. Thus, the apparent suppression of /sup 32/P incorporation due to MIX was attributable to a concurrent diminution of the specific activity of /sup 32/P in ATP. The authors conclude that increases of cAMP interfere with contraction distal to PIP/sub 2/ hydrolysis, inositol phosphate release, calcium mobilization, and enhancement of glycolysis.

  15. 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.

  16. GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation.

    PubMed Central

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

    1993-01-01

    1. The function of gamma-aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2. The GABAB agonists (-)-baclofen and 3-aminopropylphosphinic acid (3-APPA) were found to induce a dose-dependent increase of basal catecholamine secretion. The EC50s were 151 +/- 35 microM and 225 +/- 58 microM for baclofen and 3-APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mM of the specific GABAB antagonist CGP-35348. 3. In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose-dependent manner, the catecholamine secretion evoked by 10 microM nicotine and 200 microM muscimol. 4. The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose-dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol. 5. The GABAB agonists also produced a dose-dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6. The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7. The possible involvement of adenylate cyclase in the mechanism of GABAA receptor modulation of catecholamine secretion is discussed. PMID:8306105

  17. GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation.

    PubMed

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

    1993-12-01

    1. The function of gamma-aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2. The GABAB agonists (-)-baclofen and 3-aminopropylphosphinic acid (3-APPA) were found to induce a dose-dependent increase of basal catecholamine secretion. The EC50s were 151 +/- 35 microM and 225 +/- 58 microM for baclofen and 3-APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mM of the specific GABAB antagonist CGP-35348. 3. In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose-dependent manner, the catecholamine secretion evoked by 10 microM nicotine and 200 microM muscimol. 4. The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose-dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol. 5. The GABAB agonists also produced a dose-dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6. The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7. The possible involvement of adenylate cyclase in the mechanism of GABAA receptor modulation of catecholamine secretion is discussed.

  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. Cyclic AMP-dependent modulation of giant depolarizing potentials by metabotropic glutamate receptors in the rat hippocampus.

    PubMed Central

    Strata, F; Sciancalepore, M; Cherubini, E

    1995-01-01

    1. Intracellular recordings were used to study the role of metabotropic glutamate receptors (mGluRs) in modulating GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurones. 2. The mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) reduced the frequency of GDPs. The broad-spectrum ionotropic glutamate receptor antagonist kynurenic acid (1 mM) blocked GDPs. 3. In the presence of kynurenic acid, both tetanic stimulation of the hilus or bath application of quisqualic acid (1 microM) and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 20 microM) induced the appearance of GDPs. These effects were antagonized by MCPG (1 mM) or L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and blocked by bicuculline (10 microM). 4. 8-Bromo-cAMP (8-Br-cAMP, 0.3 mM), 3-isobutyl-1-methylxanthine (IBMX, 200 microM) or forskolin (30 microM) mimicked the effects of mGluR agonists on GDPs. The forskolin analogue 1,9-dideoxyforskolin (30 microM), which does not activate adenylate cyclase, was ineffective. 5. Incubation of slices in the presence of the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) (500 microM) or superfusion of Rp-cAMPS (20 microM) prevented the effects of forskolin or t-ACPD on GDPs. In the presence of kynurenic acid, the protein kinase C activator, phorbol 12,13-diacetate (2 microM) induced the appearance of GDPs. This effect was prevented by staurosporine (1 microM). However, staurosporine (1-3 microM) did not modify the effects of t-ACPD on GDPs. 6. It is suggested that, during development, mGluRs enhance the synchronous release of GABA, responsible for GDPs, through cAMP-dependent protein kinase. PMID:8583396

  20. 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.

  1. 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

  2. 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.

  3. Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders.

    PubMed

    Viollet, B; Mounier, R; Leclerc, J; Yazigi, A; Foretz, M; Andreelli, F

    2007-12-01

    In the light of recent studies in humans and rodents, AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been described as an integrator of regulatory signals monitoring systemic and cellular energy status. AMP-activated protein kinase (AMPK) has been proposed to function as a 'fuel gauge' to monitor cellular energy status in response to nutritional environmental variations. Recently, it has been proposed that AMPK could provide a link in metabolic defects underlying progression to the metabolic syndrome. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit alpha and two regulatory subunits beta and gamma. AMPK is activated by rising AMP and falling ATP. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKKbeta (calmodulin-dependent protein kinase kinase). AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischaemia playing a role in glucose transport and fatty acid oxidation. In liver, activation of AMPK results in enhanced fatty acid oxidation as well as decreased glucose production. Moreover, the AMPK system is one of the probable targets for the anti-diabetic drugs biguanides and thiazolidinediones. Thus, the relationship between AMPK activation and beneficial metabolic

  4. Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

    PubMed Central

    D'Angelo, Maximiliano A; Sanguineti, Santiago; Reece, Jeffrey M; Birnbaumer, Lutz; Torres, Héctor N; Flawiá, Mirtha M

    2004-01-01

    Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites. PMID:14556647

  5. Emergence of Escherichia coli producing extended-spectrum AmpC β-lactamases (ESAC) in animals

    PubMed Central

    Haenni, Marisa; Châtre, Pierre; Madec, Jean-Yves

    2014-01-01

    In both humans and animals, the spread of Extended-Spectrum β-Lactamases (ESBL)/AmpC producers has become a major issue, particularly due to the plasmidic dissemination of most of these genes. Besides, over-expression of the chromosomal ampC gene was largely reported in human and animal Enterobacteriaceae and, more recently, modifications within the coding region of the ampC gene [encoding Extended-spectrum AmpC β-lactamases (ESACs)] were shown to be responsible for an hydrolysis spectrum expanded to oxyiminocephalosporins in humans. In this study, among 6765 cattle E. coli isolates, 28 (0.37%) isolates harboring a reduced susceptibility to cefepime (MICs ranging from 0.5 to 12 μg/ml) were investigated as presumptive ESACs producers. Highly conserved mutations in the promoter/attenuator region were identified at positions −88, −82, −42, −18, −1, and +58. Using sequencing and cloning experiments, amino acid substitutions of the AmpC beta-lactamase were characterized at positions 287 (mostly S287N, but also S287C), 292 (A292V) and 296 (H296P), similarly to data reported in humans. Interestingly, those cattle ESAC-producing E. coli isolates predominantly belonged to the Clonal Complex (CC) 23, thus mirroring what has been described in humans. The driving forces for the selection of ESACs in animals are unknown, and their prevalence needs to be further investigated in the different animal sectors. Considering the over-representation of ESAC-producing E. coli belonging to CC23 in both humans and animals, exchanges of ESAC producers between the two populations may have occurred as well. To our best knowledge, this study is the first report of ESACs in animals worldwide, which should be considered an emerging mechanism contributing to the resistance to extended-spectrum cephalosporins in the animal population. PMID:24592257

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Synergistic effect of cAMP and palmitate in promoting altered mitochondrial function and cell death in HepG2 cells

    PubMed Central

    Zhang, Linxia; Seitz, Linsey C.; Abramczyk, Amy M.; Chan, Christina

    2009-01-01

    Saturated free fatty acids (FFAs), e.g. palmitate, have long been shown to induce toxicity and cell death in various types of cells. In this study, we demonstrate that cAMP synergistically amplifies the effect of palmitate on the induction of cell death in human hepatocellular carcinoma cell line, HepG2 cells. Elevation of cAMP level in palmitate treated cells led to enhanced mitochondrial fragmentation, mitochondrial reactive oxygen species (ROS) generation and mitochondrial biogenesis. Mitochondrial fragmentation precedes mitochondrial ROS generation and mitochondrial biogenesis, and may contribute to mitochondrial ROS overproduction and subsequent mitochondrial biogenesis. Fragmentation of mitochondria also facilitated the release of cytotoxic mitochondrial proteins, such as Smac, from the mitochondria and subsequent activation of caspases. However, cell death induced by palmitate and cAMP was caspase-independent and mainly necrotic. PMID:20026039

  14. 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

  15. 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

  16. Glucose becomes one of the worst carbon sources for E.coli on poor nitrogen sources due to suboptimal levels of cAMP.

    PubMed

    Bren, Anat; Park, Junyoung O; Towbin, Benjamin D; Dekel, Erez; Rabinowitz, Joshua D; Alon, Uri

    2016-04-25

    In most conditions, glucose is the best carbon source for E. coli: it provides faster growth than other sugars, and is consumed first in sugar mixtures. Here we identify conditions in which E. coli strains grow slower on glucose than on other sugars, namely when a single amino acid (arginine, glutamate, or proline) is the sole nitrogen source. In sugar mixtures with these nitrogen sources, E. coli still consumes glucose first, but grows faster rather than slower after exhausting glucose, generating a reversed diauxic shift. We trace this counterintuitive behavior to a metabolic imbalance: levels of TCA-cycle metabolites including α-ketoglutarate are high, and levels of the key regulatory molecule cAMP are low. Growth rates were increased by experimentally increasing cAMP levels, either by adding external cAMP, by genetically perturbing the cAMP circuit or by inhibition of glucose uptake. Thus, the cAMP control circuitry seems to have a 'bug' that leads to slow growth under what may be an environmentally rare condition.

  17. cAMP-pKA signaling regulates multiple steps of fungal infection cooperatively with Cmk1 MAP kinase in Colletotrichum lagenarium.

    PubMed

    Yamauchi, Junko; Takayanagi, Naoyuki; Komeda, Kenichi; Takano, Yoshitaka; Okuno, Tetsuro

    2004-12-01

    In Colletotrichum lagenarium, RPK1 encoding the regulatory subunit of PKA is required for pathogenicity. From the rpkl mutant that forms small colonies, we isolated three growth-suppressor mutants. All rpk1-suppressor mutants are nonpathogenic and contain amino acid changes in the PKA catalytic subunit Cpkl. To assess the roles of cyclic AMP (cAMP) signaling in detail, we generated knockout mutants of CPK1 and the adenylate cyclase gene CAC1. The cpk1 and cac1 mutants are nonpathogenic on cucumber. Interestingly, both of the mutants germinated poorly, suggesting involvement of cAMP signaling in germination. Germination defect in the cpk1 and cac1 mutants is partially rescued by incubation of the conidia at lower concentrations. Germinating conidia of the cpk1 and cac1 mutants can form appressoria, but the appressoria formed by them are nonfunctional, like those of the rpk1 mutant. Cytological analysis indicates that the appressoria of the cpk1 mutant contain larger numbers of lipid bodies compared with the wild type, whereas lipid levels in the rpk1 mutants are lower, suggesting cAMP-mediated regulation of lipid metabolism for appressorium functionality. Furthermore, the cpk1 and cacl mutants have a defect in infectious growth in plant. In C. lagenarium, Cmkl mitogen-activated protein kinase (MAPK) regulates germination, appressorium formation, and infectious growth. These results suggest that cAMP signaling controls multiple steps of fungal infection in cooperative regulation with Cmkl MAPK in C. lagenarium.

  18. MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.

    PubMed

    McManus, A M; Nielsen, K J; Marcus, J P; Harrison, S J; Green, J L; Manners, J M; Craik, D J

    1999-10-29

    MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.

  19. Glucose becomes one of the worst carbon sources for E.coli on poor nitrogen sources due to suboptimal levels of cAMP

    PubMed Central

    Bren, Anat; Park, Junyoung O.; Towbin, Benjamin D.; Dekel, Erez; Rabinowitz, Joshua D.; Alon, Uri

    2016-01-01

    In most conditions, glucose is the best carbon source for E. coli: it provides faster growth than other sugars, and is consumed first in sugar mixtures. Here we identify conditions in which E. coli strains grow slower on glucose than on other sugars, namely when a single amino acid (arginine, glutamate, or proline) is the sole nitrogen source. In sugar mixtures with these nitrogen sources, E. coli still consumes glucose first, but grows faster rather than slower after exhausting glucose, generating a reversed diauxic shift. We trace this counterintuitive behavior to a metabolic imbalance: levels of TCA-cycle metabolites including α-ketoglutarate are high, and levels of the key regulatory molecule cAMP are low. Growth rates were increased by experimentally increasing cAMP levels, either by adding external cAMP, by genetically perturbing the cAMP circuit or by inhibition of glucose uptake. Thus, the cAMP control circuitry seems to have a ‘bug’ that leads to slow growth under what may be an environmentally rare condition. PMID:27109914

  20. “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

  1. NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions.

    PubMed

    Harris, Kimberly A; Bobay, Benjamin G; Sarachan, Kathryn L; Sims, Alexis F; Bilbille, Yann; Deutsch, Christopher; Iwata-Reuyl, Dirk; Agris, Paul F

    2015-08-14

    The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis.

  2. Somatocrinin receptor coupled with cAMP-dependent protein kinase on anterior pituitary granules.

    PubMed

    Lewin, M J; Reyl-Desmars, F; Ling, N

    1983-11-01

    The molecular mechanism of growth hormone release by synthetic somatocrinin was investigated on purified hog anterior pituitary secretory granules; the granules were found to contain a cAMP-dependent protein kinase that catalyzed [gamma-32P]-ATP histone phosphorylation with maximal rates ranging from 1 to 5 nmol of Pi incorporated per mg of protein per 20 min. The activity of this enzyme was further stimulated by somatocrinin. Stimulation was observed at concentrations as low as 0.3 pM, and the half-maximal effect was obtained with 35 +/- 8 pM (n = 4). Michaelis-Menten analysis of phosphorylation kinetics suggested that the peptide did not change significantly the reaction's Vmax, but produced a dramatic increase in enzyme affinity for cAMP: the apparent Km for the nucleotide decreased from 400 X 10(-9) M under unstimulated conditions to 15 X 10(-9) M in the presence of 100 pM somatocrinin. Furthermore, a Hill plot of concentration-dependence curve indicated the existence of negative cooperativity. At the concentration of 35 pM, the less potent analogs of somatocrinin [designated hpGRF-44 to indicate source (human pancreas, hp), activity (growth hormone-releasing factor, GRF), and amino acid composition], hpGRF-(1-37) and [Phe1]hpGRF-(1-40) had 20% and 7%, respectively, of the effect of somatocrinin. The biologically inactive analog hpGRF-(2-40) had no evident effect at concentrations up to 0.1 microM. Therefore, we suggest that somatocrinin stimulation of growth hormone release involves activation of exocytosis through a phosphorylation mechanism mediated by a granular receptor coupled with a cAMP-dependent protein kinase.

  3. 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.

  4. Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.

    PubMed

    Oesch-Bartlomowicz, B; Padma, P R; Becker, R; Richter, B; Hengstler, J G; Freeman, J E; Wolf, C R; Oesch, F

    1998-07-10

    caused a marked increase in both PNP hydroxylase and NDMA demethylase. In these strains a similar db-cAMP-mediated increase was also observed in the mutation frequency, resulting from the treatment with the promutagen NDMA, which is activated by CYP2E1. Our results show that CYP2E1 in V79 cells responds in two separate ways to db-cAMP exposure depending on the amino acid residue present in the PKA recognition sequence. The enzyme is committed to a negative regulation by db-cAMP if Ser129 is the target amino acid for PKA, leading to a decrease in the metabolic activation to mutagenic and carcinogenic species. On the other hand, Ala129 or Gly129 substitution directed CYP2E1 toward a positive regulation by increasing its catalytic activities and metabolic activation to mutagenic intermediates in the presence of db-cAMP. We also obtained evidence that cAMP-mediated downregulation of wild-type (Ser129) CYP2E1 was not accompanied by its destruction but instead by its stabilization, which shows that Ser129 is not involved in CYP2E1 degradation but dictates requirements for its specific activities.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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 §...

  10. 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 §...

  11. 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 §...

  12. 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 §...

  13. 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 §...

  14. 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

  15. 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)

  16. Pulse charging of lead-acid traction cells

    NASA Astrophysics Data System (ADS)

    Smithrick, J. J.

    1980-05-01

    Pulse charging, as a method of rapidly and efficiently charging 300 amp-hour lead-acid traction cells for an electric vehicle application was investigated. A wide range of charge pulse current square waveforms were investigated and the results were compared to constant current charging at the time averaged pulse current values. Representative pulse current waveforms were: (1) positive waveform-peak charge pulse current of 300 amperes (amps), discharge pulse-current of zero amps, and a duty cycle of about 50%; (2) Romanov waveform-peak charge pulse current of 300 amps, peak discharge pulse current of 15 amps, and a duty of 50%; and (3) McCulloch waveform peak charge pulse current of 193 amps, peak discharge pulse current of about 575 amps, and a duty cycle of 94%. Experimental results indicate that on the basis of amp-hour efficiency, pulse charging offered no significant advantage as a method of rapidly charging 300 amp-hour lead-acid traction cells when compared to constant current charging at the time average pulse current value. There were, however, some disadvantages of pulse charging in particular a decrease in charge amp-hour and energy efficiencies and an increase in cell electrolyte temperature. The constant current charge method resulted in the best energy efficiency with no significant sacrifice of charge time or amp-hour output. Whether or not pulse charging offers an advantage over constant current charging with regard to the cell charge/discharge cycle life is unknown at this time.

  17. Pulse charging of lead-acid traction cells

    NASA Technical Reports Server (NTRS)

    Smithrick, J. J.

    1980-01-01

    Pulse charging, as a method of rapidly and efficiently charging 300 amp-hour lead-acid traction cells for an electric vehicle application was investigated. A wide range of charge pulse current square waveforms were investigated and the results were compared to constant current charging at the time averaged pulse current values. Representative pulse current waveforms were: (1) positive waveform-peak charge pulse current of 300 amperes (amps), discharge pulse-current of zero amps, and a duty cycle of about 50%; (2) Romanov waveform-peak charge pulse current of 300 amps, peak discharge pulse current of 15 amps, and a duty of 50%; and (3) McCulloch waveform peak charge pulse current of 193 amps, peak discharge pulse current of about 575 amps, and a duty cycle of 94%. Experimental results indicate that on the basis of amp-hour efficiency, pulse charging offered no significant advantage as a method of rapidly charging 300 amp-hour lead-acid traction cells when compared to constant current charging at the time average pulse current value. There were, however, some disadvantages of pulse charging in particular a decrease in charge amp-hour and energy efficiencies and an increase in cell electrolyte temperature. The constant current charge method resulted in the best energy efficiency with no significant sacrifice of charge time or amp-hour output. Whether or not pulse charging offers an advantage over constant current charging with regard to the cell charge/discharge cycle life is unknown at this time.

  18. The Dh gene of Drosophila melanogaster encodes a diuretic peptide that acts through cyclic AMP.

    PubMed

    Cabrero, Pablo; Radford, Jonathan C; Broderick, Kate E; Costes, Laurence; Veenstra, Jan A; Spana, Eric P; Davies, Shireen A; Dow, Julian A T

    2002-12-01

    Dh, the gene that encodes a CRF-like peptide in Drosophila melanogaster, is described. The product of this gene is a 44-amino-acid peptide (Drome-DH(44)) with a sequence almost identical to the Musca domestica and Stomoxys calcitrans diuretic hormones. There are no other similar peptides encoded within the known Drosophila genomic sequence. Functional studies showed that the deduced peptide stimulated fluid production, and that this effect was mediated by cyclic AMP in principal cells only: there was no effect on the levels of either cyclic GMP or intracellular calcium. Stimulation also elevated levels of cyclic AMP (but not cyclic GMP) phosphodiesterase, a new mode of action for this class of hormone. The transcript was localised by in situ hybridisation, and the peptide by immunocytochemistry, to two groups of three neurones in the pars intercerebralis within the brain. These cells also express receptors for leucokinin, another major diuretic peptide, implying that the cells may be important in homeostatic regulation.

  19. AMP-activated protein kinase and energy balance in breast cancer

    PubMed Central

    Zhao, Hong; Orhan, Yelda C; Zha, Xiaoming; Esencan, Ecem; Chatterton, Robert T; Bulun, Serdar E

    2017-01-01

    Cancer growth and metastasis depends on the availability of energy. Energy-sensing systems are critical in maintaining a balance between the energy supply and utilization of energy for tumor growth. A central regulator in this process is AMP-activated protein kinase (AMPK). In times of energy deficit, AMPK is allosterically modified by the binding of increased levels of AMP and ADP, making it a target of specific AMPK kinases (AMPKKs). AMPK signaling prompts cells to produce energy at the expense of growth and motility, opposing the actions of insulin and growth factors. Increasing AMPK activity may thus prevent the proliferation and metastasis of tumor cells. Activated AMPK also suppresses aromatase, which lowers estrogen formation and prevents breast cancer growth. Biguanides can be used to activate AMPK, but AMPK activity is modified by many different interacting factors; understanding these factors is important in order to control the abnormal growth processes that lead to breast cancer neoplasia. Fatty acids, estrogens, androgens, adipokines, and another energy sensor, sirtuin-1, alter the phosphorylation and activation of AMPK. Isoforms of AMPK differ among tissues and may serve specific functions. Targeting AMPK regulatory processes at points other than the upstream AMPKKs may provide additional approaches for prevention of breast cancer neoplasia, growth, and metastasis. PMID:28337254

  20. Emergence of a cefepime- and cefpirome-resistant Citrobacter freundii clinical isolate harbouring a novel chromosomally encoded AmpC beta-lactamase, CMY-37.

    PubMed

    Ahmed, Ashraf M; Shimamoto, Tadashi

    2008-09-01

    Citrobacter freundii strain 4306 was isolated from a urine specimen of a patient in March 2006 in Palestine. This strain showed a unique multidrug resistance phenotype, as it was resistant both to 7-alpha-methoxy- and oxyimino-cephalosporins, including cefepime, cefpirome and monobactams, in addition to quinolones, streptomycin and trimethoprim/sulfamethoxazole. Clavulanic acid did not act synergistically with cephalosporins by the double-disk synergy test. Molecular characterisation showed that the resistance to 7-alpha-methoxy- and oxyimino-cephalosporins was due to a novel AmpC beta-lactamase, designated CMY-37, with an isoelectric point of approximately 9.0. CMY-37 is a variant of C. freundii chromosomal AmpC enzymes with at least seven amino acid substitutions. One of these substitutions, L316I, is located within the R2 loop that is considered the hotspot region responsible for the extended substrate spectrum in class C beta-lactamases. The blaCMY-37 gene was cloned and expressed in Escherichia coli TG1. CMY-37 is chromosomally encoded and is not associated with ISEcp1-like element. Phylogenetic analysis suggested that CMY-37 is the origin of many plasmid-mediated AmpC beta-lactamases. This study highlights the emergence of cefepime and cefpirome resistance in C. freundii owing to a new type of AmpC beta-lactamase.

  1. MicroRNA-181a-mediated downregulation of AC9 protein decreases intracellular cAMP level and inhibits ATRA-induced APL cell differentiation.

    PubMed

    Zhuang, L K; Xu, G P; Pan, X R; Lou, Y J; Zou, Q P; Xia, D; Yan, W W; Zhang, Y T; Jia, P M; Tong, J H

    2014-04-10

    AC9 is one of the adenylate cyclase (AC) isoforms, which catalyze the conversion of ATP to cAMP, an important second messenger. We previously found that the integration of cAMP/PKA pathway with nuclear receptor-mediated signaling was required during all-trans retinoic acid (ATRA)-induced maturation of acute promyelocytic leukemia (APL) cells. Here we showed that AC9 could affect intracellular cAMP level and enhance the trans-activity of retinoic acid receptor. Knockdown of AC9 in APL cell line NB4 could obviously inhibit ATRA-induced differentiation. We also demonstrated that miR-181a could decrease AC9 expression by targeting 3'UTR of AC9 mRNA, finally controlling the production of intracellular cAMP. The expression of miR-181a itself could be inhibited by CEBPα, probably accounting for the differential expression of miR-181a in NB4 and ATRA-resistant NB4-R1 cells. Moreover, we found that AC9 expression was relatively lower in newly diagnosed or relapsed APL patients than in both complete remission and non-leukemia cases, closely correlating with the leukemogenesis of APL. Taken together, our studies revealed for the first time the importance of miR-181a-mediated AC9 downregulation in APL. We also suggested the potential value of AC9 as a biomarker in the clinical diagnosis and treatment of leukemia.

  2. The plasma cyclic-AMP response to noise in humans and rats—short-term exposure to various noise levels

    NASA Astrophysics Data System (ADS)

    Iwamoto, M.; Dodo, H.; Ishii, F.; Yoneda, J.; Yamazaki, S.; Goto, H.

    1988-12-01

    Rats were exposed to short-term noise which was found to activate the hypothalamohypophyseal-adrenal system and result in a decrease of adrenal ascorbic acid (AAA) and an increase of serum corticosterone (SCS). The threshold limit value lay between 60 and 70 dB(A). To characterize better the effect of noise on the human hypothalamo-hypophyseal-adrenal system, a large group of subjects was exposed to short-term noise at 85 dB(A) and higher, and tested for levels of adrenocortical steroid (cortisol) and anterior pituitary hormones such as ACTH, growth hormone (GH) and prolactin (PRL). Results in humans showed hyperfunction of the hypothalamo-pituitary system. However, as the responses in rats and humans differed, a further experiment was performed using C-AMP, a second messenger mediating many of the effects of a variety of hormones. Plasma C-AMP in humans and rats increased significantly after exposure to noise greater than 70 dB(A). We suggest that plasma C-AMP could be useful as a sensitive index for noise-related stress in the daily living environment of humans and rats.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Cyr61 Is Regulated by cAMP-Dependent Protein Kinase With Serum Levels Correlating With Prostate Cancer Aggressiveness

    PubMed Central

    Terada, Naoki; Shiraishi, Takumi; Zeng, Yu; Mooney, Steven M.; Yeater, David B.; Mangold, Leslie A.; Partin, Alan W.; Kulkarni, Prakash; Getzenberg, Robert H.

    2012-01-01

    BACKGROUND Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previously, we demonstrated that Cyr61 was highly expressed in prostate cancer (PCa) but that the expression levels were associated with a lower risk of PCa recurrence. In the present study, we demonstrate that serum Cyr61 is a potential biomarker that correlates with PCa aggressiveness. Furthermore, we also explore the potential mechanism underlying the changes in Cyr61 expression during PCa progression. METHODS Cyr61 concentrations in the medium from PCa cell lines and in serum samples obtained from PCa patients were measured by sandwich ELISA. Serum Cyr61 levels were correlated with disease characteristics and the association between Cyr61 expression changes by several types of stimulation or stress and cAMP/cAMP-dependent protein kinase (PKA) pathway were examined. RESULTS There was a positive correlation between Cyr61 levels in cell supernatants and mRNA expression in these cell lines. Serum Cyr61 levels were significantly higher in non-organ-confined PCa patients (116.3 ± 140.2 ng/ml) than in organ-confined PCa patients (79.7 ± 56.1 ng/ml) (P = 0.031). Cyr61 expression was up-regulated in response to both lysophosphatidic acid and androgen treatments which promoted PCa cell invasion. Serum starvation and phosphoinositide-3-kinase inhibition also resulted in Cyr61 up-regulation; however, they suppressed cell proliferation. Cyr61 up-regulation was correlated with an increase in cAMP and suppressed by PKA inhibition. CONCLUSIONS These findings suggest that Cyr61 expression in PCa is regulated by the cAMP/PKA pathway and that circulating Cyr61 levels are a potential serum-based biomarker for characterizing PCa. PMID:22025384

  8. Antimitogenic polymer drugs based on AMPS: monomer distribution-bioactivity relationship of water-soluble macromolecules.

    PubMed

    García-Fernández, Luis; Aguilar, María R; Fernández, María M; Lozano, Rosa M; Giménez, Guillermo; Román, Julio San

    2010-03-08

    A number of polysulfonated molecules have demonstrated their interaction with fibroblast growth factor (FGF), hampering their binding to its receptors (low affinity heparan sulfate proteoglycans (HSPG) and high affinity tyrosine kinase FGF receptors) and inhibiting the intracellular signaling and mitogenic response in cultured endothelial cells. The aim of this work was the synthesis and characterization of new copolymers based on 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with antiproliferative activity for antitumoral applications. N-Vinylpyrrolidone (VP) or butyl acrylate (BA) was copolymerized with the sulfonated monomer to obtain macromolecules with different hydrophilic/hydrophobic balance and distribution of the sulfonated groups within the macromolecules. In vitro cell culture proliferative assays showed that monomer distribution affected the inhibition of the proliferative action of FGF. Reactivity ratios of the systems were determined following the free radical copolymerization by in situ (1)H NMR, and the correlation of the monomer sequence distribution with the bioactivity is discussed.

  9. 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

  10. 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.

  11. Alumina interaction with AMPS-MPEG copolymers produced by RAFT polymerization: stability and rheological behavior.

    PubMed

    Bouhamed, H; Boufi, S; Magnin, A

    2009-05-01

    Different copolymers of 2-acrylamido-2-methylpropanesulfonic acid sodium salt (AMPS), methoxypolyethyleneglycol methacrylate (MPEG), were prepared using two methods of radical polymerization: classical and RAFT-controlled radical polymerization. The effect of polymer structure and architecture on the adsorption behavior, electrokinetic and rheological properties of the alumina suspensions was investigated. Adsorption isotherms showed that copolymer interaction depended not only on the ratio of the monomers and their distribution within the macromolecular backbone, but also on the method of copolymerization. Electrokinetic analysis indicated that adsorption of the copolymer is accompanied by a shift in the isoelectric point (IEP) towards acid pH values. Above a certain concentration, of the order of 1 wt%, the absolute value of the zeta-potential reaches a saturation plateau. At this stage, the maximum zeta-potential value (in absolute value) depends on both the ratio of the monomers for statistical copolymer and the length of the two blocks in the case of block distribution. The rheological behavior is greatly affected in the presence of added polymer; the viscosity of the alumina suspension decreases and reaches an optimum, which depends on both the ratio of the monomers and their distribution within the macromolecular backbone. The viscoelastic properties of the suspensions were found to be functions of both the structure and the architecture of the copolymer. Adding AMPS-MPEG copolymer increases the stability of the suspension via electrostatic effects, but also via steric effects induced by the polyethylene glycol (PEG) segments. The steric contribution to the stabilization process is much important in the presence of block distribution, which is more efficient as dispersant for concentrated alumina suspensions.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.

    PubMed

    Li, J; Gao, E; Mendelson, C R

    1998-02-20

    Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form

  17. The identification of novel cyclic AMP-dependent protein kinase anchoring proteins using bioinformatic filters and peptide arrays

    PubMed Central

    McLaughlin, William A.; Hou, Tingjun; Taylor, Susan S.; Wang, Wei

    2011-01-01

    A-kinase anchoring proteins (AKAPs) localize cyclic AMP-dependent protein kinase (PKA) to specific regions in the cell and place PKA in proximity to its phosphorylation targets. A computational model was created to identify AKAPs that bind to the docking/dimerization domain of the RII alpha isoform of the regulatory subunit of PKA. The model was used to search the entire human proteome, and the top candidates were tested for an interaction using peptide array experiments. Verified interactions include sphingosine kinase interacting protein and retinoic acid-induced protein 16. These interactions highlight new signaling pathways mediated by PKA. PMID:21115539

  18. Studies related to primitive chemistry. A proton and nitrogen-14 nuclear magnetic resonance amino acid and nucleic acid constituents and a and their possible relation to prebiotic

    NASA Technical Reports Server (NTRS)

    Manatt, S. L.; Cohen, E. A.; Shiller, A. M.; Chan, S. I.

    1973-01-01

    Preliminary proton nuclear magnetic resonance (NMR) studies were made to determine the applicability of this technique for the study of interactions between monomeric and polymeric amino acids with monomeric nucleic acid bases and nucleotides. Proton NMR results for aqueous solutions (D2O) demonstrated interactions between the bases cytosine and adenine and acidic and aromatic amino acids. Solutions of 5'-AMP admixed with amino acids exhibited more complex behavior but stacking between aromatic rings and destacking at high amino acids concentration was evident. The multisite nature of 5'-AMP was pointed out. Chemical shift changes for adenine and 5'-AMP with three water soluble polypeptides demonstrated that significant interactions exist. It was found that the linewidth-pH profile of each amino acid is unique. It is concluded that NMR techniques can give significant and quantitative data on the association of amino acid and nucleic acid constituents.

  19. An Investigation of the Adsorption Characteristics of 5'ATP and 5'AMP onto the Surface of Caso4 x 2H2O

    NASA Technical Reports Server (NTRS)

    Calderon, J.; Sweeney, M. A.

    1984-01-01

    A model has been proposed in which solid surfaces can act as a site for cataletic activity of condensation reactions for certain biomolecules. From this model, the adsorption characteristics of 5'ATP and 5'AMP onto the surface of CaSO4.2H2O was chosen for study. It has been proven that 5'ATP and 5'AMP do adsorb onto the surface of CaSO4. Studies were then made to determine the dependence of absorption versus time, concentration, ionic strength and pH. It was found that the adsorption of the nucleotides is highly pH dependent, primarily determined by the phosphate acid groups of the nucleic acid molecule. From this investigation, the data obtained is discussed in relation to the model for the prebiotic earth.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Effects of cold exposure on cyclic AMP concentration in plasma, liver, and brown and white adipose tissues in cold-acclimated rats

    NASA Astrophysics Data System (ADS)

    Habara, Yoshiaki

    1989-06-01

    Effects of acute cold exposure on plasma energy substrates and tissue 3',5'-adenosine monophosphate (cAMP) were analyzed in intact rats, to define an involvement of the nucleotide in nonshivering thermogenesis (NST) and resultant cold acclimation. After an acute cold exposure to -5°C, the plasma glucose level increased gradually in warm-kept control rats (C) while it decreased significantly in cold-acclimated rats (CA). However, it was increased considerably by an extreme cold exposure to -15°C in both C and CA. By contrast, plasma levels of free fatty acids (FFA) increased immediately after cold exposure and the release lasted during the period of exposure especially in C. The cold exposure also increased plasma cAMP concentration but no concomitant increase was found in the liver. In both brown (IBAT) and white (WAT) adipose tissues the nucleotide concentration showed a stepwise decrease. The observed correlation between lipolysis and plasma cAMP response after cold exposure suggests an involvement of the adenylate cyclase-cAMP system in NST via lipid metabolism, at least, in the early stages of cold acclimation.

  11. Design and membrane-disruption mechanism of charge-enriched AMPs exhibiting cell selectivity, high-salt resistance, and anti-biofilm properties.

    PubMed

    Han, Hyo Mi; Gopal, Ramamourthy; Park, Yoonkyung

    2016-02-01

    Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system, offering protection against invading pathogenic bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial and anti-biofilm properties. However, low effective stability in high-salt environments and physiological instability in biological membranes limit the applicability of naturally occurring AMPs as novel therapeutics. We therefore designed short synthetic cationic peptides by substituting key residues in myxinidin, an AMP derived from the epidermal mucus of hagfish, with lysine (Lys, K), arginine (Arg, R), and tryptophan (Trp, W). The resultant myxinidin analogs exhibited strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains, even under high-salt conditions. Moreover, these peptides showed high binding affinity for both lipopolysaccharides and lipoteichoic acids and inhibited biofilm formation by most bacteria, but did not cause significant lysis of human red blood cells and were not cytotoxic to normal human keratinocytes. Circular dichroism analysis revealed that myxinidin and its analogs assumed α-helical or β-sheet structures within artificial liposomes and bacterial membranes. In addition, bacterial killing and membrane permeation experiments demonstrated that the myxinidin analogs permeated through bacterial membranes, leading to cytoplasmic disruption and cell death. Taken together, these findings suggest myxinidin analogs may be promising candidate antibiotic agents for therapeutic application against antibiotic-resistant bacteria.

  12. 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

  13. Presence of metallo-beta-lactamases (MBL), extended-spectrum beta-lactamase (ESBL) & AmpC positive non-fermenting Gram-negative bacilli among Intensive Care Unit patients with special reference to molecular detection of blaCTX-M & blaAmpC genes

    PubMed Central

    Gupta, Richa; Malik, Abida; Rizvi, Meher; Ahmed, Moied

    2016-01-01

    Background & objectives: Non-fermenting Gram-negative bacilli (NFGNB) including Pseudomonas aeruginosa and Acinetobacter baumannii have been implicated in a variety of infections, particularly in the Intensive Care Units (ICUs). This study was aimed to overview the burden of multidrug-resistant NFGNB causing infections in ICU and also to assess the occurrence of extended-spectrum beta-lactamases (ESBLs), AmpC and metallo-beta-lactamases (MBLs) among these isolates. Methods: Bacterial culture, identification and antibiotic susceptibility were carried out. ESBLs and AmpC were detected both phenotypically and genotypically. MBL was detected by modified Hodge and imipenem-ethylenediaminetetraacetic acid double-disc synergy test. Results: NFGNB represented 45 (37%) of total 121 Gram negative isolates. Multidrug resistance was observed in 66.9 per cent and 72.5 per cent isolates of P. aeruginosa and A. baumannii, respectively. Detection by phenotypic methods showed presence of ESBL, AmpC and MBL in 21.4, 51.1 and 21.4 per cent isolates, respectively. When detected genotypically by polymerase chain reaction, ESBL and AmpC were detected in 21.4 and 41.4 per cent of NFGNB isolates, respectively. BlaCTX-M (21.4%) was the most prevalent gene responsible for ESBL production. Interpretation & conclusions: Most of the NFGNB isolated from ICU patients were multidrug-resistant and producers of ESBL, AmpC and MBL. A regular surveillance is required to detect ESBL, AmpC and MBL producers, especially in ICU patients. PMID:27934808

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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…

  1. 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.

  2. 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

  3. Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.

    PubMed

    Baichoo, N; Heyduk, T

    1999-03-01

    CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830-10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA]2-), iron complexed to ethylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine mesylate ([Fe-HDFO]+), and copper complexed to o-phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA] and [Fe-DTPA]2- of CRP-cAMP detected hypersensitivities in the DNA-binding F alpha-helix, the interdomain hinge, and the ends of the C alpha-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA]2- also detected reductions in cleavage in the D and E alpha-helices, which are involved in DNA recognition. Cleavage by [Fe-HDFO]+ of CRP-cAMP detected hypersensitivities in beta-strand 8, the B alpha-helix, as well as in parts of the F and C alpha-helices. [Fe-HDFO]+ also detected protections from cleavage in beta-strands 4 to 5 and their intervening loop, beta-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E alpha-helices. Cleavage by [(OP)2Cu]+ of CRP-cAMP detected hypersensitivities in beta-strands 9 and 11 as well as in the D and E alpha-helices. [(OP)2Cu]+ also detected protections in the C alpha-helix , the interdomain hinge, and beta-strands 2-7. Binding of cGMP to CRP resulted in changes in

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Chemical Synthesis of a 5'-Terminal TMG-Capped Triribonucleotide m(3)(2,2,7)G(5)(')pppAmpUmpA of U1 RNA.

    PubMed

    Sekine, Mitsuo; Kadokura, Michinori; Satoh, Takahiko; Seio, Kohji; Wada, Takeshi; Fischer, Utz; Sumpter, Vicki; Lührmann, Reinhard

    1996-06-26

    The 5'-terminal TMG-capped triribonucleotide, m(3)(2,2,7)G(5)(')pppAmpUmpA, has been synthesized by condensation of an appropriately protected triribonucleotide derivative of ppAmpUmpA with a new TMG-capping reagent. During this total synthesis, it was found that the regioselective 2'-O-methylation of 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N-(4-monomethoxytrityl)adenosine was achieved by use of MeI/Ag(2)O without affecting the base moiety. A new route to 2-N,2-N-dimethylguanosine from guanosine via a three-step reaction has also been developed by reductive methylation using paraformaldehyde and sodium cyanoborohydride. These key intermediates were used as starting materials for the construction of a fully protected derivative of pAmpUmpA and a TMG-capping reagent of Im-pm(3)(2,2,7)G. The target TMG-capped tetramer, m(3)(2,2,7)G(5)(')pppAmpUmpA, was synthesized by condensation of a partially protected triribonucleotide 5'-terminal diphosphate species, ppA(MMTr)mpUmpA, with Im-pm(3)(2,2,7)G followed by treatment with 80% acetic acid. The structure of m(3)(2,2,7)G(5)(')pppAmpUmpA was characterized by (1)H and (31)P NMR spectroscopy as well as enzymatic assay using snake venom phosphodiesterase, calf intestinal phosphatase, and nuclease P1.

  19. Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter

    PubMed Central

    Moscoso, Joana A.; Schramke, Hannah; Tosi, Tommaso; Dehbi, Amina; Jung, Kirsten

    2015-01-01

    ABSTRACT Nucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory

  20. Properties of Starch-Poly(acrylamide-co-2-acrylamido-2-methylpropanesulfonic acid) Graft Copolymers Prepared by Reactive Extrusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graft copolymers of starch with acrylamide and 2-acrylamido-2-methylpropanesulfonic acid (AMPS) were prepared by reactive extrusion in a twin-screw extruder. The weight ratio of total monomer to starch was fixed at 1:3, while the molar fraction of AMPS in the monomer feed ranged from 0 to 0.119. Mon...

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Dispersion of alumina suspension using comb-like and diblock copolymers produced by RAFT polymerization of AMPS and MPEG.

    PubMed

    Bouhamed, H; Boufi, S; Magnin, A

    2007-08-15

    Different copolymers of 2-acrylamido-2-methylpropanesulfonic acid sodium salt (AMPS) methoxypolyethyleneglycol methacrylate (MPEG) with statistical and diblock distributions were prepared using RAFT-controlled radical polymerization. The effect of polymer architecture and monomer ratio on the adsorption behavior, electrokinetic, and stability properties of the alumina suspensions was investigated. Adsorption isotherms showed that copolymer interaction depended on both the ratio of the monomers and their distribution within the macromolecular backbone. Changes in the electrokinetic properties of the alumina suspension after addition of the copolymers were investigated by monitoring the particle zeta-potential as a function of pH. A continuous shift in the isoelectric point IEP to a more acidic value was observed and particle charges were reversed when the amount of copolymer added exceeded a critical level.

  15. 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.

  16. Rapid Induction of Neural Differentiation in Human Umbilical Cord Matrix Mesenchymal Stem Cells by cAMP-elevating Agents

    PubMed Central

    Shahbazi, Atefeh; Safa, Majid; Alikarami, Fatemeh; Kargozar, Saeid; Asadi, Mohammad Hossein; Joghataei, Mohammad Taghi; Soleimani, Mansoureh

    2016-01-01

    Human umbilical cord matrix (hUCM) is considered as a promising source of mesenchymal stem cells (MSCs) due to several advantages over other tissues. The potential of neural differentiation of hUCM-MSCs is of great interest in the context of treating neurodegenerative diseases. In recent years, considerable efforts have been made to establish in vitro conditions for improving the differentiation of hUCM-MSCs toward neuronal cells. In the present study, we evaluated the neural differentiation potential of hUCM-MSCs in the presence of cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). hUCM-MSCs were isolated from fetal umbilical cord and characterized by flow cytometry analysis for mesenchymal specific markers. Mesodermal differentiation potential was assessed through selective media with lineage-specific induction factors. For assessment of neural differentiation, cells were cultured in the presence of cAMP-elevating agents for 8 and 24 h. The neuronal differentiated MSCs were characterized for neuronal specific markers by immunocytochemistry and western blotting. Isolated hUCM-MSCs were found positive for mesenchymal markers (CD73, CD90, and CD105) while negative for hematopoietic markers (CD34 and CD45) .Following neural induction, most cells represented neural-like cells morphology. Neural markers including β-tubulin III (Tuj-1), neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP-2) and nestin were expressed in treated cells with respect to control group. The astrocyte specific marker, glial fibrillary acidic protein (GFAP) was also shown by immunofluorescence in treated cells. (These findings demonstrate that hUCM-MSCs have the ability to rapidly differentiate into neural cell types of neuron-like cells and astrocytes by cAMP-elevating agents without the presence of growth factors. PMID:27942503

  17. Rapid Induction of Neural Differentiation in Human Umbilical Cord Matrix Mesenchymal Stem Cells by cAMP-elevating Agents.

    PubMed

    Shahbazi, Atefeh; Safa, Majid; Alikarami, Fatemeh; Kargozar, Saeid; Asadi, Mohammad Hossein; Joghataei, Mohammad Taghi; Soleimani, Mansoureh

    2016-01-01

    Human umbilical cord matrix (hUCM) is considered as a promising source of mesenchymal stem cells (MSCs) due to several advantages over other tissues. The potential of neural differentiation of hUCM-MSCs is of great interest in the context of treating neurodegenerative diseases. In recent years, considerable efforts have been made to establish in vitro conditions for improving the differentiation of hUCM-MSCs toward neuronal cells. In the present study, we evaluated the neural differentiation potential of hUCM-MSCs in the presence of cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). hUCM-MSCs were isolated from fetal umbilical cord and characterized by flow cytometry analysis for mesenchymal specific markers. Mesodermal differentiation potential was assessed through selective media with lineage-specific induction factors. For assessment of neural differentiation, cells were cultured in the presence of cAMP-elevating agents for 8 and 24 h. The neuronal differentiated MSCs were characterized for neuronal specific markers by immunocytochemistry and western blotting. Isolated hUCM-MSCs were found positive for mesenchymal markers (CD73, CD90, and CD105) while negative for hematopoietic markers (CD34 and CD45) .Following neural induction, most cells represented neural-like cells morphology. Neural markers including β-tubulin III (Tuj-1), neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP-2) and nestin were expressed in treated cells with respect to control group. The astrocyte specific marker, glial fibrillary acidic protein (GFAP) was also shown by immunofluorescence in treated cells. (These findings demonstrate that hUCM-MSCs have the ability to rapidly differentiate into neural cell types of neuron-like cells and astrocytes by cAMP-elevating agents without the presence of growth factors.

  18. Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Mediation by adenosine 3':5'-monophosphate.

    PubMed

    Drezner, M K; Eisenbarth, G S; Neelon, F A; Lebovitz, H E

    1975-02-13

    The effects of growth hormone-dependent serum factors on amino acid transport and on cartilage cyclic AMP levels in embryonic chicken cartilage were studied in vitro. Cartilages incubated in medium containing rat serum showed a significantly greater uptake of alpha-amino [1-14C] isobutyrate or [1-14C] cycloleucine than control cartilages incubated in medium alone. Normal rat serum (5%) added to the incubation medium also caused an increase in cartilage cyclic AMP content (from as little as 23% to as much as 109%). The factors in serum which increase cartilage cyclic AMP and amino acid uptake are growth hormone dependent, since neither growth hormone itself nor serum from hypophysectomized rats restores these serum factors. Studies comparing the ability of sera with varying amounts of growth hormone-dependent factors to stimulate amino-aminoisobutyrate transport and to increase cartilage cyclic AMP show a striking linear correlation between the two effects (r=0.977). Theophylline and prostaglandin E1, WHICH RAISE CARTILAGE CYCLIC AMP also increase amino-aminoisobutyrate transport. Exogenous cyclic AMP, N6-monobutyryl cyclic AMP and n6, 02'-dibutyryl cyclic AMP increase cartilage amino-aminoisobutyrate transport. The data are compatible with the thesis that growth hormone-dependent serum factors increase cartilage amino acid transport by elevating cartilage cyclic AMP.

  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. Rewiring global regulator cAMP receptor protein (CRP) to improve E. coli tolerance towards low pH.

    PubMed

    Basak, Souvik; Geng, Hefang; Jiang, Rongrong

    2014-03-10

    Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we tried to improve strain acidic tolerance from its transcription level, i.e. we adopted error-prone PCR method to engineer global regulator cAMP receptor protein (CRP) of Escherichia coli to improve its performance at low pH. The best mutant AcM1 was identified from random mutagenesis libraries based on its growth performance. AcM1 almost doubled (0.113h(-1)) the growth rate of the control (0.062h(-1)) at pH 4.24. It also demonstrated better thermotolerance than the control at 48°C, whose growth was completely inhibited at this temperature. Quantitative real time reverse transcription PCR results revealed a stress response overlap among low pH stress-, oxidative stress- and osmotic stress-related genes. The chief enzyme responsible for cell acid tolerance, glutamate decarboxylase, demonstrated over twofold activity in AcM1 compared to the control. Differential binding properties of AcM1 mutant CRP with Class-I, II, and III CRP-dependent promoters suggested that modifications to native CRP may lead to transcription profile changes. Hence, we believe that transcriptional engineering of global regulator CRP can provide a new strain engineering alternative for E. coli.

  1. 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.

  2. 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…

  3. 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.

  4. 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...

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

    PubMed

    Christensen, Henriette L; Păunescu, Teodor G; Matchkov, Vladimir; Barbuskaite, Dagne; Brown, Dennis; Damkier, Helle H; Praetorius, Jeppe

    2017-01-01

    The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H(+)-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na(+)-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na(+) amounted to <10% of the pHi recovery rate observed in the presence of Na(+) Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

  14. Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

    PubMed

    Li, Jiaxin; Dou, Xiaobing; Li, Songtao; Zhang, Ximei; Zeng, Yong; Song, Zhenyuan

    2015-11-01

    Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation.

  15. cAMP/protein kinase A activates cystic fibrosis transmembrane conductance regulator for ATP release from rat skeletal muscle during low pH or contractions.

    PubMed

    Tu, Jie; Lu, Lin; Cai, Weisong; Ballard, Heather J

    2012-01-01

    We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTR(inh)-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTR(inh)-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na(+)/H(+)-exchanger (NHE) with amiloride or the Na(+)/Ca(2+)-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  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. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. cAMP receptor protein (CRP)-mediated resistance/tolerance in bacteria: mechanism and utilization in biotechnology.

    PubMed

    Geng, Hefang; Jiang, Rongrong

    2015-06-01

    Cyclic AMP receptor protein (CRP) is one of the seven global regulators in Escherichia coli, which regulates the expression of over 490 genes. It contains a cAMP binding N-terminal domain and a DNA binding C-terminal domain, connected via a short hinge region. Various stress-tolerant E. coli mutants had been obtained through transcriptional engineering of CRP. This review aims to shed some light on the possible mechanism behind these CRP variants, from the change in CRP structure, transcription profile, and DNA binding affinity. The amino acid substitutions are distributed along the protein-certain mutations have shown higher frequency than others, such as T127N and D138Y. β-Galactosidase reporter gene assay revealed that CRP mutants had lower binding affinity with all three classes of CRP-dependent promoters as compared to native CRP, which probably would change cellular transcription profile. Different CRP mutants would induce different cellular transcription profile in E. coli, but there are common genes differentially expressed in these variants, including upregulated gadAB and downregulated nontransporter genes aspA and tnaA, and transporter/poringenes malE, mglB, cstA, and lamB. We believe that transcriptional engineering of CRP can provide an alternative strain engineering method for E. coli and its detailed mechanism may need further investigations.

  8. Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development.

    PubMed Central

    Hopkinson, S B; Pollenz, R S; Drummond, I; Chisholm, R L

    1989-01-01

    We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat. Images PMID:2555685

  9. 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…

  10. 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

  11. 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.

  12. Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism

    SciTech Connect

    Farese, R.V.; Sabir, M.A.; Larson, R.E.

    1981-12-01

    We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of (3H)glycerol and (14C)palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.

  13. Regulation of the Src homology 2 domain-containing inositol 5'-phosphatase (SHIP1) by the cyclic AMP-dependent protein kinase.

    PubMed

    Zhang, Jun; Walk, Scott F; Ravichandran, Kodi S; Garrison, James C

    2009-07-24

    Many agents that activate hematopoietic cells use phos pha tidyl ino si tol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) to initiate signaling cascades. The SH2 domain-containing inositol 5' phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phos pha tidyl ino si tol 3-kinase and reducing the levels of PtdIns 3,4,5-P(3). Activation of the cyclic AMP-de pend ent protein kinase (PKA) also opposes many of the pro-inflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phos pho ryl a tion with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phos pho ryl a ted by PKA to a stoichiometry of 0.6 mol of PO(4)/mol of SHIP1. In (32)P-labeled HEK-293 cells transfected with SHIP1, stimulation with Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) or activation of the beta-adrenergic receptor increased the phos pho ryl a tion state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phos pho ryl a tion of SHIP1. Phosphorylation of SHIP1 in vitro or in cells by PKA increased the 5' phosphatase activity of SHIP1 by 2-3-fold. Elevation of Ca(2+) in DT40 cells in response to B cell receptor cross-linking, an indicator of PtdIns 3,4,5-P(3) levels, was markedly blunted by pretreatment with Sp-cAMPS. This effect was absent in SHIP(-/-) DT40 cells showing that the effect of Sp-cAMPS in DT40 cells is SHIP1-de pend ent. Sp-cAMPS also blunted the ability of the B cell receptor to increase the phos pho ryl a tion of Akt in DT40 and A20 cells. Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phosphorylated and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation.

  14. Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds

    PubMed Central

    Marín-Aguilar, Fabiola; Pavillard, Luis E.; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D.

    2017-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases. PMID:28146060

  15. Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

    The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K+ channels, Na+ channels, Ca2+ release activated Ca2+ channels, Cl- channels, gap junctional channels, glucose carriers, Na+/H+-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na+/Ca2+-exchanger, H+-ATPase and Na+/K+-ATPase. AMPK activates ubiquitin ligase Nedd4–2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3β (GSK3β). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-κB. All those cellular effects serve to regulate transport proteins. PMID:24366036

  16. A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase.

    PubMed

    Xu, Z-X; Liang, J; Haridas, V; Gaikwad, A; Connolly, F P; Mills, G B; Gutterman, J U

    2007-11-01

    Avicins, a family of plant triterpene electrophiles, can trigger apoptosis-associated tumor cell death, and suppress chemical-induced carcinogenesis by its anti-inflammatory, anti-mutagenic, and antioxidant properties. Here, we show that tumor cells treated with benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone, an apoptosis inhibitor, and Bax(-/-)Bak(-/-) apoptosis-resistant cells can still undergo cell death in response to avicin D treatment. We demonstrate that this non-apoptotic cell death is mediated by autophagy, which can be suppressed by chloroquine, an autophagy inhibitor, and by specific knockdown of autophagy-related gene-5 (Atg5) and Atg7. Avicin D decreases cellular ATP levels, stimulates the activation of AMP-activated protein kinase (AMPK), and inhibits mammalian target of rapamycin (mTOR) and S6 kinase activity. Suppression of AMPK by compound C and dominant-negative AMPK decreases avicin D-induced autophagic cell death. Furthermore, avicin D-induced autophagic cell death can be abrogated by knockdown of tuberous sclerosis complex 2 (TSC2), a key mediator linking AMPK to mTOR inhibition, suggesting that AMPK activation is a crucial event targeted by avicin D. These findings indicate the therapeutic potential of avicins by triggering autophagic cell death.

  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. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. Metabolic flux analysis of Arthrobacter sp. CGMCC 3584 for cAMP production based on 13C tracer experiments and gas chromatography-mass spectrometry.

    PubMed

    Niu, Huanqing; Chen, Yong; Yao, Shiwei; Liu, Lixia; Yang, Chen; Li, Bingbing; Liu, Dong; Xie, Jingjing; Chen, Xiaochun; Wu, Jinglan; Ying, Hanjie

    2013-12-01

    Arthrobacter sp. CGMCC 3584 are able to produce cAMP from glucose by the purine synthesis pathway via de novo or salvage biosynthesis. In order to gain an improved understanding of its metabolism, (13)C-labeling experiment and gas chromatography-mass spectrometry (GC-MS) analysis were employed to determine the metabolic network structure and estimate the intracellular fluxes. GC-MS analysis helps to reflect the activity of the intracellular pathways and reactions. The metabolic network mainly contains glycolytic and pentose phosphate pathways, the tricarboxylic acid cycle, and the inactive glyoxylate shunt. Hypoxanthine as a precursor of cAMP and sodium fluoride as an inhibitor of glycolysis were found to increase the cAMP production, as well as the flux through the PP pathway. The effects of adding hypoxanthine and sodium fluoride are discussed based on the enzyme assays and metabolic flux analysis. In conclusion, our results provide quantitative insights into how cells manipulate the metabolic network under different culture conditions and this may be of value in metabolic regulation for desirable production.

  13. 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.

  14. 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

  15. In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

    PubMed Central

    Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian

    2014-01-01

    To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections. PMID:24982064

  16. In vitro and in vivo activities of antimicrobial peptides developed using an amino acid-based activity prediction method.

    PubMed

    Wu, Xiaozhe; Wang, Zhenling; Li, Xiaolu; Fan, Yingzi; He, Gu; Wan, Yang; Yu, Chaoheng; Tang, Jianying; Li, Meng; Zhang, Xian; Zhang, Hailong; Xiang, Rong; Pan, Ying; Liu, Yan; Lu, Lian; Yang, Li

    2014-09-01

    To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections.

  17. 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.

  18. 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.

  19. 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.

  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. A Ric8/synembryn homolog promotes Gpa1 and Gpa2 activation to respectively regulate cyclic AMP and pheromone signaling in Cryptococcus neoformans.

    PubMed

    Gong, Jinjun; Grodsky, Jacob D; Zhang, Zhengguang; Wang, Ping

    2014-10-01

    The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1(Q284L) allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1(Q284L) negatively affected its interaction with Ric8, whereas the activated Gpa2(Q203L) allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans.

  6. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

    SciTech Connect

    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi; Fujita, Norihisa

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  7. A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

    PubMed Central

    Gong, Jinjun; Grodsky, Jacob D.; Zhang, Zhengguang

    2014-01-01

    The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans. PMID:25084863

  8. Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products.

    PubMed

    Fuentes, Eduardo; Palomo, Iván

    2013-01-01

    Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists.

  9. Relationship between Platelet PPARs, cAMP Levels, and P-Selectin Expression: Antiplatelet Activity of Natural Products

    PubMed Central

    Fuentes, Eduardo; Palomo, Iván

    2013-01-01

    Platelets are no longer considered simply as cells participating in thrombosis. In atherosclerosis, platelets are regulators of multiple processes, with the recruitment of inflammatory cells towards the lesion sites, inflammatory mediators release, and regulation of endothelial function. The antiplatelet therapy has been used for a long time in an effort to prevent and treat cardiovascular diseases. However, limited efficacy in some patients, drug resistance, and side effects are limitations of current antiplatelet therapy. In this context, a large number of natural products (polyphenols, terpenoids, alkaloids, and fatty acids) have been reported with antiplatelet activity. In this sense, the present paper describes mechanisms of antiplatelet action of natural products on platelet P-selectin expression through cAMP levels and its role as peroxisome proliferator-activated receptors agonists. PMID:24324520

  10. 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.

  11. Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance.

    PubMed

    Zhang, Hongfang; Chong, Huiqing; Ching, Chi Bun; Jiang, Rongrong

    2012-05-01

    The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor protein (CRP) of Escherichia coli can improve cell phenotypes. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. The target phenotype in this study is strain osmotolerance. Amino acid mutations were introduced to CRP by either error-prone PCR or DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1-MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1-MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to the wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9 M NaCl, the growth rate of MT6 is 0.113 h(-1), while that of WT is 0.077 h(-1). MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose, and sucrose. DNA microarray analysis showed that genes involved in colanic acid biosynthesis are up-regulated in the absence of salt stress, whereas carbohydrate metabolic genes are differentially expressed under NaCl stress when comparing MT6 to WT. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. Exploring sequence requirements for C3/C4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β-lactamase

    PubMed Central

    Drawz, Sarah M; Taracila, Magdalena; Caselli, Emilia; Prati, Fabio; Bonomo, Robert A

    2011-01-01

    In Pseudomonas aeruginosa, the chromosomally encoded class C cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C3/C4 carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C3/C4 carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C3/C4 β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C3/C4 β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C3/C4 carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme. PMID:21404358

  18. 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.

  19. [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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.