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Sample records for ectonucleoside triphosphate diphosphohydrolases

  1. Cloning, molecular characterization and expression of ecto-nucleoside triphosphate diphosphohydrolase-1 from Torpedo electric organ.

    Science.gov (United States)

    Martín-Satué, Mireia; Torrejón-Escribano, Benjamín; Felipe, Antonio; de Aranda, Inmaculada Gómez; Elías, Marc; Marsal, Jordi; Blasi, Juan; Solsona, Carles

    2007-01-01

    During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.

  2. Kinetic characterization of ecto-nucleoside triphosphate diphosphohydrolases in brain nerve terminals during rat postnatal development

    Science.gov (United States)

    Stanojević, I.; Drakulić, D.; Petrović, S.; Milošević, M.; Jovanović, N.; Horvat, A.

    2011-12-01

    A family of enzymes named ecto-nucleoside triphosphate diphosphohydrolase (NTPDases) catalyzes the termination of ATP and ADP actions. Three different NTPDases (NTPDase 1-3), differing in their preference for a substrate, have been localized in the brain of adult mammals. The goal of our study was to clarify ATP and ADP hydrolyzing activities and kinetic parameters of NTPDases in synaptic plasma membranes (SPM) isolated from 15-, 30-, 60- and 90-days-old female rat brains. ATP and ADP hydrolysis were maximal in the presence of Mg2+ and showed insensitivity to ion-transporting ATPase inhibitors. The pronounced increase in both, ATP and ADP hydrolysis, were found in the SPM isolated from rats in the first month of life, stayed at the same level in the second month, and then decreased in adulthood. Kinetic analysis are also developmental-dependent, and together with the rate of ATP:ADP hydrolysis, point that all three NTPDases are present in SPM isolated from different developmental stages, with different, developmental-dependent proportion of activities. The lowest velocity and the highest affinity were observed for ATP hydrolyses, while the highest velocity and lowest affinity were detected for ADP hydrolyses in SPM isolated from 15-day old rats. Since specific ATP and ADP hydrolysis were lowest in this stage, we concluded that velocity is crucial for ATPase-, while affinity is for ADPase-part of NTPDases. Increased NTPDases activities, changes in their hydrolysis velocity and substrates affinities during rat postnatal development indicate involvement of adenine nucleotides in processes implicated to neuronal maturation and augmented neuroprotection.

  3. Influence of Ecto-nucleoside triphosphate diphosphohydrolase activity on Trypanosoma cruzi infectivity and virulence.

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    Ramon F Santos

    Full Text Available BACKGROUND: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. METHODOLOGY/PRINCIPAL FINDINGS: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin, or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. CONCLUSIONS/SIGNIFICANCE: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target

  4. Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence.

    Science.gov (United States)

    Sansom, Fiona M; Riedmaier, Patrice; Newton, Hayley J; Dunstone, Michelle A; Müller, Christa E; Stephan, Holger; Byres, Emma; Beddoe, Travis; Rossjohn, Jamie; Cowan, Peter J; d'Apice, Anthony J F; Robson, Simon C; Hartland, Elizabeth L

    2008-05-09

    Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.

  5. Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications

    Directory of Open Access Journals (Sweden)

    Kiss David S

    2009-04-01

    Full Text Available Abstract Background Based on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3 may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR hypothalamic structures in the rat brain, here we investigated: 1. The cellular and subcellular localization of NTPDase3; 2. The effects of 17β-estradiol on the expression level of hypothalamic NTPDase3; and 3. The effects of NTPDase inhibition in hypothalamic synaptosomal preparations. Methods Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode. Results Combined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD indicated that γ-amino-butyric-acid- (GABA ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of

  6. Pregnancy enhances the sensitivity of glomerular ecto-adenosine triphosphate-diphosphohydrolase to products of activated polymorphonuclear leukocytes

    NARCIS (Netherlands)

    Faas, MM; Bakker, WW; Baller, JFW; Schuiling, GA

    To test the hypothesis that pregnancy enhances the sensitivity of glomerular ecto-adenosine triphosphate-diphosphohydrolase to products of activated polymorphonuclear leukocytes, cryostat-cut kidney sections of pregnant and cycling rats were exposed to activated polymorphonuclear leukocytes and

  7. Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

    Science.gov (United States)

    Summers, Emma L; Cumming, Mathew H; Oulavallickal, Tifany; Roberts, Nicholas J; Arcus, Vickery L

    2017-08-01

    Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life. © 2017 The Protein Society.

  8. Structural insight into activation mechanism of Toxoplasma gondii nucleoside triphosphate diphosphohydrolases by disulfide reduction.

    Science.gov (United States)

    Krug, Ulrike; Zebisch, Matthias; Krauss, Michel; Sträter, Norbert

    2012-01-27

    The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258-268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys(258)-Cys(268) show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg(2+) to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg(492) and Glu(493) in NTPDase1 by glycines in NTPDase3.

  9. Characterization of nucleoside triphosphate diphosphohydrolase activity in Trichomonas gallinae and the influence of penicillin and streptomycin in extracellular nucleotide hydrolysis.

    Science.gov (United States)

    Borges, Fernanda Pires; de Brum Vieira, Patrícia; Wiltuschnig, Renata C M; Tasca, Tiana; De Carli, Geraldo Attilio; Bonan, Carla Denise

    2008-06-01

    Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.

  10. Inhibition of rat synaptic membrane Na⁺/K⁺-ATPase and ecto-nucleoside triphosphate diphosphohydrolases by 12-tungstosilicic and 12-tungstophosphoric acid.

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    Čolović, Mirjana B; Bajuk-Bogdanović, Danica V; Avramović, Nataša S; Holclajtner-Antunović, Ivanka D; Bošnjaković-Pavlović, Nada S; Vasić, Vesna M; Krstić, Danijela Z

    2011-12-01

    The in vitro influence of Keggin structure polyoxotungstates, 12-tungstosilicic acid, H(4)SiW(12)O(40) (WSiA) and 12-tungstophosphoric acid, H(3)PW(12)O(40) (WPA), and monomer Na(2)WO(4) × 2H(2)O on rat synaptic plasma membrane (SPM) Na(+)/K(+)-ATPase and E-NTPDase activity was studied, whereas the commercial porcine cerebral cortex Na(+)/K(+)-ATPase served as a reference. Dose-dependent Na(+)/K(+)-ATPase inhibition was obtained for all investigated compounds. Calculated IC(50) (10 min) values, in mol/l, for SPM/commercial Na(+)/K(+)-ATPase, were: 3.4 × 10(-6)/4.3 × 10(-6), 2.9 × 10(-6)/3.1 × 10(-6) and 1.3 × 10(-3)/1.5 × 10(-3) for WSiA, WPA and Na(2)WO(4) × 2H(2)O, respectively. In the case of E-NTPDase, increasing concentrations of WSiA and WPA induced its activity reduction, while Na(2)WO(4) × 2H(2)O did not noticeably affect the enzyme activity at all investigated concentrations (up to 1 × 10(-3)mol/l). IC(50) (10 min) values, obtained from the inhibition curves, were (in mol/l): 4.1 × 10(-6) for WSiA and 1.6 × 10(-6) for WPA. Monolacunary Keggin anion was found as the main active molecular species present under physiological conditions (in the enzyme assays, pH 7.4), for the both polyoxotungstates solutions (1 mmol/l), using Fourier transform infrared (FT-IR) and micro-Raman spectroscopy. Additionally, commercial porcine cerebral cortex Na(+)/K(+)-ATPase was exposed to the mixture of Na(2)WO(4) × 2H(2)O and WSiA at different concentrations. Additive inhibition effect was achieved for lower concentrations of Na(2)WO(4) × 2H(2)O/WSiA (≤ 1 × 10(-3)/4 × 10(-6) mol/l), while antagonistic effect was obtained for all higher concentrations of the inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Rat pancreas secretes particulate ecto-nucleotidase CD39

    DEFF Research Database (Denmark)

    Sørensen, Christiane Elisabeth; Amstrup, Jan; Rasmussen, Hans N

    2003-01-01

    -ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization...

  12. ATP release, generation and hydrolysis in exocrine pancreatic duct cells

    DEFF Research Database (Denmark)

    Kowal, Justyna Magdalena; Yegutkin, G.G.; Novak, Ivana

    2015-01-01

    Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our...... dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting...

  13. Aggregation of rat neutrophils by nucleotide triphosphates.

    OpenAIRE

    Ford-Hutchinson, A. W.

    1982-01-01

    1 Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) at concentrations of 3 x 10(-7)M and greater cause a rapid partially reversible aggregation of rat polymorphonuclear leucocytes. 2 Other nucleotide phosphates are much less active at producing aggregation responses; the agonist potencies being UTP greater than ATP greater than guanosine 5'-triphosphate, cytidine 5'-triphosphate, thymidine 5'-triphosphate; ATP greater than adenosine 5'-diphosphate (ADP) greater than adenosine ...

  14. Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

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    A.M. Kettlun

    2000-07-01

    Full Text Available Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10 and Desirée (ATPase/ADPase = 1 isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

  15. Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro.

    Science.gov (United States)

    Caiazzo, Elisabetta; Tedesco, Idolo; Spagnuolo, Carmela; Russo, Gian Luigi; Ialenti, Armando; Cicala, Carla

    2016-06-01

    Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP--and ADPase activity in rat platelets.

  16. Chemoelectrical energy conversion of adenosine triphosphate

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    Sundaresan, Vishnu Baba; Sarles, Stephen Andrew; Leo, Donald J.

    2007-04-01

    Plant and animal cell membranes transport charged species, neutral molecules and water through ion pumps and channels. The energy required for moving species against established concentration and charge gradients is provided by the biological fuel - adenosine triphosphate (ATP) -synthesized within the cell. The adenosine triphosphatase (ATPases) in a plant cell membrane hydrolyze ATP in the cell cytoplasm to pump protons across the cell membrane. This establishes a proton gradient across the membrane from the cell exterior into the cell cytoplasm. This proton motive force stimulates ion channels that transport nutrients and other species into the cell. This article discusses a device that converts the chemical energy stored in adenosine triphosphate into electrical power using a transporter protein, ATPase. The V-type ATPase proteins used in our prototype are extracted from red beet(Beta vulgaris) tonoplast membranes and reconstituted in a bilayer lipid membrane or BLM formed from POPC and POPS lipids. A pH7 medium that can support ATP hydrolysis is provided on both sides of the membrane and ATP is dissolved in the pH7 buffer on one side of the membrane. Hydrolysis of ATP results in the formation of a phosphate ion and adenosine diphosphate. The energy from the reaction activates ATPase in the BLM and moves a proton across the membrane. The charge gradient established across the BLM due to the reaction and ion transport is converted into electrical current by half-cell reference electrodes. The prototype ATPase cell with an effective BLM area of 4.15 mm2 carrying 15 μl of ATPase proteins was observed to develop a steady state peak power output of 70 nW, which corresponds to a specific power of 1.69 μW/cm2 and a current density of 43.4 μA/cm2 of membrane area.

  17. Stability of [5-3H]uridine-5'-triphosphate

    International Nuclear Information System (INIS)

    Brabec, D.

    1980-01-01

    The effect of temperature, the solvent systems, evaporation, volume reduction, etc. on the decomposition rate of [5- 3 H]uridine-5'-triphosphate was investigated. The decomposition rates and optimum storage conditions were established. The possibility of reducing the duration of the purification and separation process was examined. (author)

  18. Synthesis of adenosine triphosphate tritiated in position 2 and 8

    International Nuclear Information System (INIS)

    Cossery, Jean-Michel

    1986-01-01

    Adenosine triphosphate or ATP is an important molecule present at the cellular level in many fundamental biochemical mechanism, and the study of its metabolism is therefore of particular interest. In this thesis for pharmacy graduation, the author first describes the different steps of synthesis and purification leading to chloride-2-ATP, a precursor of the final tritiated molecule. Then, the author explains the tritiation of this molecule to obtain an ATP tritiated in position 2 and in position 8 [fr

  19. Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase

    International Nuclear Information System (INIS)

    Porta, Jason; Kolar, Carol; Kozmin, Stanislav G.; Pavlov, Youri I.; Borgstahl, Gloria E. O.

    2006-01-01

    X-ray crystallographic analysis of human inosine triphosphate pyrophosphohydrolase provided the secondary structure and active-site structure at 1.6 Å resolution in an orthorhombic crystal form. The structure gives a framework for future structure–function studies employing site-directed mutagenesis and for the identification of substrate/product-binding sites. The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 Å resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix α1, the loop before α6 and helix α7 to cap off the active site when substrate is bound

  20. Where is electronic energy stored in adenosine triphosphate?

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    Arabi, Alya A; Matta, Chérif F

    2009-04-09

    The gas-phase electronic energy of the hydrolysis of methyl triphosphate, a model of adenosine 5'-triphosphate (ATP), is partitioned into local (atomic and group) contributions. A modified definition of Lipmann's "group transfer potential" is proposed on the basis of the partitioning of the total electronic energy into atomic contributions within the framework of the quantum theory of atoms in molecules (QTAIM). The group transfer potential is defined here as the sum of the atomic energies forming the group in ATP minus the sum of the energies of the same atoms in inorganic phosphate. It is found that the transfer potential of the terminal phosphate group in ATP is significantly reduced, from +241.7 to +73.1 kcal/mol, as a result of complexation with magnesium. This is accompanied by a concomitant change in the energy of reaction from -168.6 to -24.9 kcal/mol. Regions within ATP where the electronic energy changes the most upon hydrolysis are identified. The study is conducted at the DFT/B3LYP/6-31+G(d,p) level of theory.

  1. A terbium(III)-organic framework for highly selective sensing of cytidine triphosphate.

    Science.gov (United States)

    Zhao, Xi Juan; He, Rong Xing; Li, Yuan Fang

    2012-11-21

    Highly selective sensing of cytidine triphosphate (CTP) against other triphosphate nucleosides including ATP, GTP and UTP is successfully achieved with a luminescent terbium(III)-organic framework (TbOF) of [Tb(2)(2,3-pzdc)(2)(ox)(H(2)O)(2)](n) (2,3-pzdc(2-) = 2,3-pyrazinedicarboxylate, ox(2-) = oxalate).

  2. Gram-Scale Chemical Synthesis of Base-Modified Ribonucleoside-5'-O-Triphosphates.

    Science.gov (United States)

    Shanmugasundaram, Muthian; Senthilvelan, Annamalai; Kore, Anilkumar R

    2016-12-01

    This unit delineates a simple, reliable, straight-forward, general, and efficient chemical method for the synthesis of modified nucleoside-5'-O-triphosphates such as 5-methylcytidine-5'-O-triphosphate (5-Me-CTP), pseudouridine-5'-O-triphosphate (pseudo-UTP), and N 1 -methylpseudouridine-5'-O-triphosphate (N 1 -methylpseudo-UTP), starting from the corresponding nucleoside. The reaction utilizes an improved protection-free "one-pot, three-step" Ludwig synthetic strategy that involves the monophosphorylation of the nucleoside with phosphorous oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding ribonucleoside triphosphate (NTP) in moderate yields. It is noteworthy that the reaction affords high purity (>99.5%) NTPs after DEAE Sepharose column purification. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  3. Enzymatic synthesis of adenosine-5'-[γ-33P]triphosphate

    International Nuclear Information System (INIS)

    Hanker, I.

    1990-01-01

    In certain conditions, incubation of ''carrier-free'' H 3 33 PO 4 with ADP in the presence of a mixture of enzymes gave rise to [γ- 33 P]ATP in a yield better than 98%. The product can be used for commercial purposes or, without separation, applied directly to enzymatic synthesis of a number of nucleoside-5'-[α- 33 P]triphosphates. In some cases, however, the product was formed in low yields. It is suggested that failures in the application of commercial enzymes were due to the inhibiting effect of ammonium sulfate. The specificity of labelling in the gamma position was commonly higher than 95%. (author). 4 figs., 5 tabs., 6 refs

  4. Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39.

    Science.gov (United States)

    Zhong, Xiaotian; Buddha, Madhavan; Guidotti, Guido; Kriz, Ron; Somers, Will; Mosyak, Lidia

    2008-11-01

    CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P3(2), with unit-cell parameters a = b = 118.1, c = 81.6 A and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 A data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P3(2) lattice and rigid-body refined and position-minimized with PHENIX.

  5. On the structure of thorium and americium adenosine triphosphate complexes

    International Nuclear Information System (INIS)

    Mostapha, Sarah; Berton, Laurence; Boubals, Nathalie; Zorz, Nicole; Charbonnel, Marie-Christine; Fontaine-Vive, Fabien; Den Auwer, Christophe; Solari, Pier Lorenzo

    2014-01-01

    The actinides are chemical poisons and radiological hazards. One challenge to better appraise their toxicity and develop countermeasures in case of exposure of living organisms is to better assess pathways of contamination. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, nucleotides and in particular adenosine triphosphate nucleotide (ATP) may be considered critical target building blocks for actinides. Combinations of spectroscopic techniques (Fourier transformed Infra Red [FTIR], Electro-spray Ionization Mass Spectrometry [ESI-MS], and Extended X-ray Absorption Fine Structure [EXAFS]) with quantum chemical calculations have been implemented in order to assess the actinides coordination arrangement with ATP. We describe and compare herein the interaction of ATP with thorium and americium; thorium(IV) as a representative of actinide(IV) like plutonium(IV) and americium(III) as a representative of all heavier actinides. In the case of thorium, an insoluble complex is readily formed. In the case of americium, a behavior identical to that described previously for lutetium has been observed with insoluble and soluble complexes. The comparative study of ATP complexation with Th(IV) and Am(III) shows their ability to form insoluble complexes for which a structural model has been proposed by analogy with previously described Lu(III) complexes. (authors)

  6. On the structure of thorium and americium adenosine triphosphate complexes.

    Science.gov (United States)

    Mostapha, Sarah; Fontaine-Vive, Fabien; Berthon, Laurence; Boubals, Nathalie; Zorz, Nicole; Solari, Pier Lorenzo; Charbonnel, Marie Christine; Den Auwer, Christophe

    2014-11-01

    The actinides are chemical poisons and radiological hazards. One challenge to better appraise their toxicity and develop countermeasures in case of exposure of living organisms is to better assess pathways of contamination. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, nucleotides and in particular adenosine triphosphate nucleotide (ATP) may be considered critical target building blocks for actinides. Combinations of spectroscopic techniques (Fourier transformed Infra Red [FTIR], Electrospray Ionization Mass Spectrometry [ESI-MS], and Extended X-ray Absorption Fine Structure [EXAFS]) with quantum chemical calculations have been implemented in order to assess the actinides coordination arrangement with ATP. We describe and compare herein the interaction of ATP with thorium and americium; thorium(IV) as a representative of actinide(IV) like plutonium(IV) and americium(III) as a representative of all heavier actinides. In the case of thorium, an insoluble complex is readily formed. In the case of americium, a behavior identical to that described previously for lutetium has been observed with insoluble and soluble complexes. The comparative study of ATP complexation with Th(IV) and Am(III) shows their ability to form insoluble complexes for which a structural model has been proposed by analogy with previously described Lu(III) complexes.

  7. Adenosine triphosphate-magnesium dichloride during hyperdynamic porcine endotoxemia: Effects on hepatosplanchnic oxygen exchange and metabolism

    NARCIS (Netherlands)

    Asfar, Pierre; Nalos, Marek; Pittner, Antje; Theisen, Marc; Ichai, Carole; Ploner, Franz; Georgieff, Michael; Ince, Can; Brückner, Uwe Bernd; Leverve, Xavier Maurice; Radermacher, Peter; Froeba, Gebhard

    2002-01-01

    OBJECTIVE: To assess the effects of adenosine triphosphate-magnesium dichloride (ATP-MgCl2) on systemic and hepatosplanchnic hemodynamics, oxygen exchange, and energy metabolism over 24 hrs of hyperdynamic normotensive porcine endotoxemia. DESIGN: Prospective, randomized, controlled experimental

  8. Synthesis of high specific activity tritium labelled [2-3H]-adenosine-5'-triphosphate

    International Nuclear Information System (INIS)

    Jaiswal, D.K.; Morimoto, H.; Trump, E.L.; Williams, P.G.; Wemmer, D.E.

    1996-01-01

    A procedure for high level tritium labelling at the C2-H position of adenosine 5'-triphosphate ([2- 3 H]-ATP, 1), based on the tritiodehalogenation reaction of 2-bromoadenosine 5'-triphosphate (2) has been elaborated. This precursor was prepared in a six-step synthesis from guanosine. The tritiodehalogenation of (2) for three hours over palladium oxide in phosphate buffer yielded tritium labelled ATP with high specific activity, in good chemical yield. (author)

  9. NTPDase5/PCPH as a New Target in Highly Aggressive Tumors: A Systematic Review

    Directory of Open Access Journals (Sweden)

    Paula Andreghetto Bracco

    2014-01-01

    Full Text Available The protooncogene PCPH was recently identified as being the ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5. This protooncogene is converted into an oncogene by a single base pair deletion, resulting in frame change and producing a premature stop codon, leading to a mutated protein (mt-PCPH with only 27 kDa, which is much smaller than the original 47 kDa protein. Overexpression of the PCPH as well as the mutated PCPH increases the cellular resistance to stress and apoptosis. This is intriguing considering that the active form, that is, the oncogene, is the mutated PCPH. Several studies analyzed the expression of NTPDase5/mt-PCPH in a wide range of tumor cells and evaluated its role and mechanisms in cancer and other pathogenic processes. The main point of this review is to integrate the findings and proposed theories about the role played by NTPDase5/mt-PCPH in cancer progression, considering that these proteins have been suggested as potential early diagnostic tools and therapy targets.

  10. NTPDase2+ cells generate lingual epithelia and papillae

    Directory of Open Access Journals (Sweden)

    Li eFeng

    2012-11-01

    Full Text Available The tongue epithelium is one of the most rapidly self-renewing tissues in adult mammals. Multiple stem cell populations are currently believed to exist in tongue epithelia. Keratin 14 (K14 positive cells differentiate into either lingual epithelia or lingual papillae, while ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2 is associated with neural stem cells and astrocyte-like cells ensheathing the migrating neuroblasts. Here, using a transgenic mouse expressing rtTA from the mouse NTPDase2 promoter, we generated an inducible model by treatment with Doxycycline. By immunohistochemical analysis and in situ hybridization, we found exclusive expression of NTPDase2 in lingual epithelia and lingual papillae. Using inducible genetic cell fate mapping, we further showed that the NTPDase2+ cells generated lingual papillae and epithelia in the adult tongue. Finally, building on our previously proposed paradigm of cell migration stream, a model is further described here for lingual epithelia cell genesis. In short, the current results not only extend our understanding of the cell migration stream in lingual epithelia and lingual papillae, but they also support the concept of multiple stem cell populations in lingual epithelia and papillae.

  11. Sepsis induced by cecal ligation and perforation (CLP) alters nucleotidase activities in platelets of rats.

    Science.gov (United States)

    Pereira, Renata S; Bertoncheli, Claudia M; Adefegha, Stephen A; Castilhos, Lívia G; Silveira, Karine L; Rezer, João Felipe P; Doleski, Pedro H; Abdalla, Fátima H; Santos, Karen F; Leal, Claudio A M; Santos, Roberto C V; Casali, Emerson A; Moritz, Cesar E J; Stainki, Daniel R; Leal, Daniela B R

    2017-10-01

    Sepsis is a potentially lethal condition, and it is associated with platelet alterations. The present study sought to investigate the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA) in the platelets of rats that were induced with sepsis. Male Wistar rats were divided into three groups of ten animals each: a negative control group (normal; NC); a group that underwent surgical procedures (sham); and a group that underwent cecal ligation and perforation (CLP). The induction of sepsis was confirmed by bacteremia, and the causative pathogen identified was Escherichia coli. Hematological parameters showed leukocytosis and thrombocytopenia in animals in the septic group. The results also revealed that there were significant (p CLP group compared to the sham and control groups. Conversely, ADP hydrolysis was significantly decreased (p CLP group compared to the sham and control groups. Purine levels were analyzed by high-performance liquid chromatography (HPLC) in serum samples from control, sham, and CLP groups. Increased concentrations of ATP, adenosine, and inosine were found in the CLP group compared to the sham and control groups. Conversely, the concentrations of ADP and AMP in the CPL group were not significantly altered. We suggest that alterations in hematological parameters, nucleotide hydrolysis in platelets, and nucleotide concentrations in serum samples of rats with induced sepsis may be related to thromboembolic events. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; De Carli, Geraldo Attilio; Bonan, Carla Denise; Tasca, Tiana

    2012-03-01

    Trichomonas vaginalis is a parasite from the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. The neutrophil infiltration has been considered to be primarily responsible for cytological changes observed at infection site, and the chemoattractants can play an important role in this leukocytic recruitment. Nitric oxide (NO) is one of the most widespread mediator compounds, and it is implicated in modulation of immunological mechanisms. Extracellular nucleotides and nucleosides are signaling molecules involved in several processes, including immune responses and control of leukocyte trafficking. Ectonucleoside triphosphate diphosphohydrolase members, ecto-5'-nucleotidase, and adenosine deaminase (ectoADA) have been characterized in T. vaginalis. Herein, we investigated the effects of purinergic system on NO production by neutrophils stimulated with T. vaginalis. The trophozoites were able to induce a high NO synthesis by neutrophils through iNOS pathway. The extracellular nucleotides ATP, ADP, and ATPγS (a non-hydrolyzable ATP analog) showed no significant change in NO secretion. In contrast, adenosine and its degradation product, inosine, promoted a low production of the compound. The immunosuppressive effect of adenosine upon NO release by neutrophils occurred due to adenosine A(2A) receptor activation. The ecto-5'-nucleotidase activity displayed by T. vaginalis was shown to be important in adenosine generation, indicating the efficiency of purinergic cascade. Our data suggest the influence of purinergic signaling, specifically adenosinergic system, on NO production by neutrophils in T. vaginalis infection, contributing to the immunological aspects of disease.

  13. ATP-consuming and ATP-generating enzymes secreted by pancreas

    DEFF Research Database (Denmark)

    Yegutkin, Gennady G; Samburski, Sergei S; Jalkanen, Sirpa

    2006-01-01

    Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim of this st......Pancreatic acini release ATP in response to various stimuli, including cholecystokinin octapeptide (CCK-8), as we show in the present study. There were indications that pancreatic juice also contains enzymes that could hydrolyze ATP during its passage through the ductal system. The aim...... of this study was to determine which ATP-degrading and possibly ATP-generating enzymes were present in pancreatic secretion. For this purpose, pancreatic juice was collected from anesthetized rats stimulated with infusion of CCK-8. Purine-converting activities in juice samples were assayed by TLC using either...... [gamma-(32)P]ATP or (14)C/(3)H-labeled and unlabeled nucleotides as appropriate substrates. Data show that the juice contains the enzyme ecto-nucleoside triphosphate diphosphohydrolase that can hydrolyze both [(14)C]ATP and [(3)H]ADP about equally well, i.e. CD39. Reverse-phase high-performance liquid...

  14. Preservation of cochlear function in Cd39 deficient mice.

    Science.gov (United States)

    Vlajkovic, Srdjan M; Housley, Gary D; Thorne, Peter R; Gupta, Rita; Enjyoji, Keiichi; Cowan, Peter J; Charles Liberman, M; Robson, Simon C

    2009-07-01

    Signalling actions of extracellular nucleotides via P2 receptors influence cellular function in most tissues. In the inner ear, P2 receptor signaling is involved in many processes including the regulation of hearing sensitivity and the cochlea's response to noise stress. CD39 (NTPDase1/ENTPD1) is an ectonucleotidase (ecto-nucleoside triphosphate diphosphohydrolase) that can hydrolyse purine and pyrimidine nucleoside tri- and di-phosphates to generate monophosphate nucleosides. Mice null for Cd39 exhibit major alterations in haemostasis and profound alterations in inflammatory and thrombotic reactions. Studies in the cochlea have suggested the involvement of purinergic-type signals that could be modulated by CD39 in regulation of cochlear blood flow and also auditory neurotransmission. This study aimed to determine the auditory phenotype of adult Cd39 null mice on the C57BL6 background. Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were unaffected in Cd39-deficient mice across the range of test frequencies, suggesting normal neural and outer hair cell function. Mutant mice also showed little difference to wild type mice in vulnerability to acoustic trauma. Gene expression analysis of other membrane-bound NTPDases with comparable hydrolytic activity demonstrated an up-regulation of Entpd2 and Entpd8 in the cochleae of Cd39 deficient mice. These findings suggest that Cd39 deletion alone does not adversely affect cochlear function, possibly as compensatory up-regulation of other surface located NTPDases may offset predicted alterations in cochlear homeostasis.

  15. Adenosine triphosphate-binding cassette transporters mediate chemokine (C-C motif) ligand 2 secretion from reactive astrocytes: relevance to multiple sclerosis pathogenesis

    NARCIS (Netherlands)

    Kooij, G.; Mizee, M.R.; van Horssen, J.; Reijerkerk, A.; Witte, M.E.; Drexhage, J.A.R.; van der Pol, SM; Van Het Hof, B; Scheffer, G.L.; Scheper, R.J.; Dijkstra, C.D.; van der Valk, P.; de Vries, H.E.

    2011-01-01

    Adenosine triphosphate-binding cassette efflux transporters are highly expressed at the blood-brain barrier and actively hinder passage of harmful compounds, thereby maintaining brain homoeostasis. Since, adenosine triphosphate-binding cassette transporters drive cellular exclusion of potential

  16. Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

    NARCIS (Netherlands)

    Verschuur, A. C.; van Gennip, A. H.; Muller, E. J.; Voûte, P. A.; Vreken, P.; van Kuilenburg, A. B.

    1999-01-01

    Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood

  17. Inosine triphosphate pyrophosphatase and thiopurine s-methyltransferase genotypes relationship to azathioprine-induced myelosuppression

    NARCIS (Netherlands)

    Zelinkova, Zuzana; Derijks, Luc J. J.; Stokkers, Pieter C. F.; Vogels, Esther W. M.; van Kampen, Antoine H. C.; Curvers, Wouter L.; Cohn, Danny; van Deventer, Sander J. H.; Hommes, Daniël W.

    2006-01-01

    BACKGROUND & AIMS: The use of azathioprine (AZA) in inflammatory bowel disease (IBD) patients is limited by toxicity, which occurs in up to 20% of treated patients. Mutations in the thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) genes have been associated with

  18. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Bajaj, Mamta; Moriyama, Hideaki

    2007-01-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V M of 1.8 Å 3 Da −1

  19. Purification, crystallization and preliminary crystallographic analysis of deoxyuridine triphosphate nucleotidohydrolase from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Bajaj, Mamta [School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States); Moriyama, Hideaki, E-mail: hmoriyama2@unl.edu [Department of Chemistry, e-Toxicology and Biotechnology, University of Nebraska-Lincoln, Hamilton Hall, Lincoln, Nebraska 68588-0304 (United States); School of Biological Sciences, University of Nebraska-Lincoln, Manter Hall, Lincoln, Nebraska 68588-0304 (United States)

    2007-05-01

    The first crystallization of deoxyuridine triphosphate nucleotidohydrolase from plant, Arabidopsis thaliana, has been performed. An additive, taurine, was effective in producing the single crystal. The deoxyuridine triphosphate nucleotidohydrolase gene from Arabidopsis thaliana was expressed and the gene product was purified. Crystallization was performed by the hanging-drop vapour-diffusion method at 298 K using 2 M ammonium sulfate as the precipitant. X-ray diffraction data were collected to 2.2 Å resolution using Cu Kα radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 69.90, b = 70.86 Å, c = 75.55 Å. Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a V{sub M} of 1.8 Å{sup 3} Da{sup −1}.

  20. Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39

    International Nuclear Information System (INIS)

    Zhong, Xiaotian; Buddha, Madhavan; Guidotti, Guido; Kriz, Ron; Somers, Will; Mosyak, Lidia

    2008-01-01

    The ecto-enzymatic domain of rat E-NTPDase1 CD39 was expressed and purified and diffraction-quality crystals of the enzyme were obtained. CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P3 2 , with unit-cell parameters a = b = 118.1, c = 81.6 Å and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 Å data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P3 2 lattice and rigid-body refined and position-minimized with PHENIX

  1. Estimation of adenosine triphosphate utilization of rat mast cells during and after anaphylactic histamine secretion

    DEFF Research Database (Denmark)

    Johansen, Torben

    1990-01-01

    Determination of the cellular content of adenosine triphosphate (ATP) and the rate of ATP-synthesis were used to estimate the cellular utilization of ATP in relation to anaphylactic histamine secretion. There was an increased rate of oxidative ATP-synthesis and a decreased cellular ATP content...... during the time period of histamine secretion and immediately after its completion. During secretion the additional ATP-utilization above the basal level of ATP-synthesis was 0.51 pmol/10(3) cells. 2.5 min after cell activation, the rate of additional ATP-utilization was 0.30 pmol/10(3) cells...

  2. The immediate nucleotide precursor, guanosine triphosphate, in the riboflavin biosynthetic pathway

    International Nuclear Information System (INIS)

    Mitsuda, Hisateru; Nakajima, Kenji; Nadamoto, Tomonori

    1977-01-01

    In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashbyii. The added purines, at 10 -4 M, were effectively incorporated into riboflavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the course of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed. (auth.)

  3. Adenosine Triphosphate-Sensitive Potassium Currents in Heart Disease and Cardioprotection.

    Science.gov (United States)

    Nichols, Colin G

    2016-06-01

    The subunit makeup of the family of adenosine triphosphate-sensitive potassium channel (KATP) channels is more complex and labile than thought. The growing association of Kir6.1 and SUR2 variants with specific cardiovascular electrical and contractile derangements and the clear association with Cantu syndrome establish the importance of appropriate activity in normal function of the heart and vasculature. Further studies of such patients will reveal new mutations in KATP subunits and perhaps in proteins that regulate KATP synthesis, trafficking, or location, all of which may ultimately benefit therapeutically from the unique pharmacology of KATP channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Signal transduction events induced by extracellular guanosine 5?triphosphate in excitable cells

    OpenAIRE

    Pietrangelo, T.; Guarnieri, S.; Fulle, S.; Fan?, G.; Mariggi?, M. A.

    2006-01-01

    A better understanding of the physiological effects of guanosine-based purines should help clarify the complex subject of purinergic signalling. We studied the effect of extracellular guanosine 5?triphosphate (GTP) on the differentiation of two excitable cell lines that both have specific binding sites for GTP: PC12 rat pheochromocytoma cells and C2C12 mouse skeletal muscle cells. PC12 cells can be differentiated into fully functional sympathetic-like neurons with 50?00 ng ml?1 of nerve growt...

  5. Measurement of adenosine triphosphate (ATP) content in single red blood cells using the firefly bioluminescent reaction

    Energy Technology Data Exchange (ETDEWEB)

    Kostuk, R.K.; Muhs, A.G.; Kirkpatrick, F.H.; Gabel, C.W.

    1977-01-01

    A unique optical instrument is described which uses the firefly bioluminscent reaction to measure adenosine triphosphate (ATP) levels in single red blood cells. The method allows chemical content level to be associated with individual cell features. The optical instrument consists of a phase contrast microscope to view cells, a pulsed argon-ion laser to rupture the cell membrane, and a photon counting system to measure the bioluminescent yield. The technique has been calibrated against a standard ATP measurement using bulk analysis methods. The ATP loss mechanism for blood cells in a controlled depletion experiment was also investigated.

  6. Effects of adenosine triphosphate concentration on motor force regulation during skeletal muscle contraction

    Science.gov (United States)

    Wei, J.; Dong, C.; Chen, B.

    2017-04-01

    We employ a mechanical model of sarcomere to quantitatively investigate how adenosine triphosphate (ATP) concentration affects motor force regulation during skeletal muscle contraction. Our simulation indicates that there can be negative cross-bridges resisting contraction within the sarcomere and higher ATP concentration would decrease the resistance force from negative cross-bridges by promoting their timely detachment. It is revealed that the motor force is well regulated only when ATP concentration is above a certain level. These predictions may provide insights into the role of ATP in regulating coordination among multiple motors.

  7. Effect of extracellular adenosine triphosphate on activity of osteoblast like cells - biomed 2013.

    Science.gov (United States)

    Mehta, Siddhant K; Tucci, Michelle A; Benghuzzi, Hamed A

    2013-01-01

    Platelet dense granules contain serotonin, adenosine triphosphate (ATP), and adenosine diphosphate (ADP). These molecules are present in platelet rich plasma (PRP), and may therefore have an impact on the efficacy of PRP therapy. Additionally, nucleotides are important extracellular signaling molecules in a variety of tissue types including bone. The purpose of this investigation was to evaluate the in vitro dose-dependent effects of extracellular adenosine triphosphate (ATP) exposure on activity of human osteoblast-like cells. MG-63 cells were exposed to phosphate buffered saline (control group) or ATP solution (20 µM, 100µM, 200 µM). Osteoblast viability was evaluated at 24, 48, and 72 hours using nonspecific and osteoblast-specific markers and cellular morphology. No significant differences in total protein, malonlydialdehyde (MDA), or glutathione were observed with ATP exposure at any timepoint. High dose ATP exposure resulted in a significantly higher production of nitric oxide compared to controls and other groups. With respect to alkaline phosphatase activity and osteopontin production, no significant differences were present with ATP exposure. Overall conclusion: Extracellular ATP exposure modulated osteoblast activity with no change in cell viability in vitro.

  8. Determination of adenosine disodium triphosphate using prulifloxacin-terbium(III) as a fluorescence probe by spectrofluorimetry

    International Nuclear Information System (INIS)

    Yu Fengshan; Li Lin; Chen Fang

    2008-01-01

    A new spectrofluorimetric method is developed for determination of adenosine disodium triphosphate (ATP). The interactions between prulifloxacin (PUFX)-Tb 3+ complex and adenosine disodium triphosphate has been studied by using UV-vis absorption and fluorescence spectra. Using prulifloxacin-Tb 3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the prulifloxacin-Tb 3+ complex at λ = 545 nm and the enhanced fluorescence intensity is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 4.0 x 10 -7 to 2.0 x 10 -5 mol L -1 , and the detection limit (3 σ/k) is 1.7 x 10 -8 mol L -1 . This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in real pharmaceutical samples. The mechanism of fluorescence enhancement of prulifloxacin-Tb 3+ complex by ATP was also discussed

  9. Agonist action of adenosine triphosphates at the human P2Y1 receptor.

    Science.gov (United States)

    Palmer, R K; Boyer, J L; Schachter, J B; Nicholas, R A; Harden, T K

    1998-12-01

    The agonist selectivity for adenosine di- and triphosphates was determined for the human P2Y1 receptor stably expressed in human 1321N1 astrocytoma cells and was studied under conditions in which nucleotide metabolism was both minimized and assessed. Cells were grown at low density on glass coverslips, encased in a flow-through chamber, and continuously superfused with medium, and Ca2+ responses to nucleotides were quantified. Superfusion with high performance liquid chromatographically purified ADP, ATP, 2-methylthio-ADP, and 2-methylthio-ATP resulted in rapid Ca2+ responses, with EC50 values of 10 +/- 5, 304 +/- 51, 2 +/- 1, and 116 +/- 50 nM, respectively. Similar peak responses were observed with maximal concentrations of these four agonists and with the hydrolysis-resistant adenine nucleoside triphosphate adenosine-5'-O-(3-thiotriphosphate). No conversion of [3H]ATP to [3H]ADP occurred under these conditions. Similar full agonist activities of ATP, 2-methylthio-ATP, and ADP were observed in human embryonic kidney 293 cells, which natively express the P2Y1 receptor. In contrast to these results, Leon et al. [FEBS Lett 403:26-30 (1997)] and Hechler et al. [Mol Pharmacol 53:727-733 (1998)] recently reported that, whereas ADP and 2-methylthio-ADP were agonists, ATP and 2-methylthio-ATP were weak antagonists in studies of the human P2Y1 receptor expressed in human Jurkat cells. To assess whether differences in the degree of receptor reserve might explain this discrepancy of results, P2Y1 receptor-expressing 1321N1 cells were incubated for 24 hr with adenosine-5'-O-(2-thiodiphosphate), with the goal of down-regulating the level of functional receptors. Pretreatment with adenosine-5'-O-(2-thiodiphosphate) resulted in a 10-fold rightward shift in the concentration-effect curve for ADP; in contrast, the agonist activity of ATP was completely abolished. Taken together, our results indicate that adenosine di- and triphosphates are agonists at the human P2Y1 receptor

  10. Localization of RNA transcription sites in insect oocytes using microinjections of 5-bromouridine 5'-triphosphate.

    Directory of Open Access Journals (Sweden)

    Dmitry Bogolyubov

    2007-06-01

    Full Text Available In the present study we used 5-bromouridine 5'-triphosphate (BrUTP microinjections to localize the transcription sites in oocytes of insects with different types of the ovarium structure: panoistic, meroistic polytrophic, and meroistic telotrophic. We found that in an insect with panoistic ovaries (Acheta domesticus, oocyte nuclei maintain their transcription activity during the long period of oocyte growth. In insects with meroistic ovaries (Tenebrio molitor and Panorpa communis, early oocyte chromosomes were found to be transcriptionally active, and some transcription activity still persist while the karyosphere, a compact structure formed by all condensed oocyte chromosomes, begins to develop. At the latest stages of karyosphere development, no anti-Br-RNA signal was registered in the karyosphere.

  11. Adenosine triphosphate-guided pulmonary vein isolation for atrial fibrillation: the UNmasking Dormant Electrical Reconduction by Adenosine TriPhosphate (UNDER-ATP) trial.

    Science.gov (United States)

    Kobori, Atsushi; Shizuta, Satoshi; Inoue, Koichi; Kaitani, Kazuaki; Morimoto, Takeshi; Nakazawa, Yuko; Ozawa, Tomoya; Kurotobi, Toshiya; Morishima, Itsuro; Miura, Fumiharu; Watanabe, Tetsuya; Masuda, Masaharu; Naito, Masaki; Fujimoto, Hajime; Nishida, Taku; Furukawa, Yoshio; Shirayama, Takeshi; Tanaka, Mariko; Okajima, Katsunori; Yao, Takenori; Egami, Yasuyuki; Satomi, Kazuhiro; Noda, Takashi; Miyamoto, Koji; Haruna, Tetsuya; Kawaji, Tetsuma; Yoshizawa, Takashi; Toyota, Toshiaki; Yahata, Mitsuhiko; Nakai, Kentaro; Sugiyama, Hiroaki; Higashi, Yukei; Ito, Makoto; Horie, Minoru; Kusano, Kengo F; Shimizu, Wataru; Kamakura, Shiro; Kimura, Takeshi

    2015-12-07

    Most of recurrent atrial tachyarrhythmias after pulmonary vein isolation (PVI) for atrial fibrillation (AF) are due to reconnection of PVs. The aim of the present study was to evaluate whether elimination of adenosine triphosphate (ATP)-induced dormant PV conduction by additional energy applications during the first ablation procedure could reduce the incidence of recurrent atrial tachyarrhythmias. We randomly assigned 2113 patients with paroxysmal, persistent, or long-lasting AF to either ATP-guided PVI (1112 patients) or conventional PVI (1001 patients). The primary endpoint was recurrent atrial tachyarrhythmias lasting for >30 s or those requiring repeat ablation, hospital admission, or usage of Vaughan Williams class I or III antiarrhythmic drugs at 1 year with the blanking period of 90 days post ablation. Among patients assigned to ATP-guided PVI, 0.4 mg/kg body weight of ATP provoked dormant PV conduction in 307 patients (27.6%). Additional radiofrequency energy applications successfully eliminated dormant conduction in 302 patients (98.4%). At 1 year, 68.7% of patients in the ATP-guided PVI group and 67.1% of patients in the conventional PVI group were free from the primary endpoint, with no significant difference (adjusted hazard ratio [HR] 0.89; 95% confidence interval [CI] 0.74-1.09; P = 0.25). The results were consistent across all the prespecified subgroups. Also, there was no significant difference in the 1-year event-free rates from repeat ablation for any atrial tachyarrhythmia between the groups (adjusted HR 0.83; 95% CI 0.65-1.08; P = 0.16). In the catheter ablation for AF, we found no significant reduction in the 1-year incidence of recurrent atrial tachyarrhythmias by ATP-guided PVI compared with conventional PVI. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  12. Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate)

    NARCIS (Netherlands)

    Hensgens, CMH; Santos, H; Zhang, CH; Kruizinga, WH; Hansen, TA

    1996-01-01

    Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the

  13. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

    Science.gov (United States)

    Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

    2017-11-01

    5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely re...

  15. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    Science.gov (United States)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  16. Effect of caffeine, caffeic acid and their various combinations on enzymes of cholinergic, monoaminergic and purinergic systems critical to neurodegeneration in rat brain-In vitro.

    Science.gov (United States)

    Akomolafe, S F; Akinyemi, A J; Ogunsuyi, O B; Oyeleye, S I; Oboh, G; Adeoyo, O O; Allismith, Y R

    2017-09-01

    Caffeine and caffeic acid are two bioactive compounds that are present in plant foods and are major constituent of coffee, cocoa, tea, cola drinks and chocolate. Although not structurally related, caffeine and caffeic acid has been reported to elicit neuroprotective properties. However, their different proportional distribution in food sources and possible effect of such interactions are not often taken into consideration. Therefore, in this study, we investigated the effect of caffeine, caffeic acid and their various combinations on activities of some enzymes [acetylcholinesterase (AChE), monoamine oxidase (MAO) ecto-nucleoside triphosphate diphosphohydrolase (E-NTPase), ecto-5 1 -nucleotidase (E-NTDase) and Na + /K + ATPase relevant to neurodegeneration in vitro in rat brain. The stock concentration of caffeine and caffiec acid and their various proportional combinations were prepared and their interactions with the activities of these enzymes were assessed (in vitro) in different brain structures. The Fe 2+ and Cu 2+ chelating abilities of the samples were also investigated. The results revealed that caffeine, caffeic acid and their various combinations exhibited inhibitory effect on activities of AChE, MAO, E-NTPase and E-NTDase, but stimulatory effect on Na + /K + ATPase activity. The combinations also exhibited Fe 2+ and Cu 2+ chelating abilities. Considering the various combinations, a higher caffeine to caffeic acid ratio produced significantly highest enzyme modulatory effects; these were significantly lower to the effect of caffeine alone but significantly higher than the effect of caffeic acid alone. These findings may provide new insight into the effect of proportional combination of these bioactive compounds as obtained in many foods especially with respect to their neuroprotective effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord [v1; ref status: indexed, http://f1000r.es/3rm

    Directory of Open Access Journals (Sweden)

    Eric McCoy

    2014-07-01

    Full Text Available Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides.  Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP to adenosine in primary somatosensory neurons.  Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons.  Ectonucleoside triphosphate diphosphohydrolases (ENTPDs comprise a class of enzymes that dephosphorylate extracellular ATP and ADP.  Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3 was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG, in the dorsal horn of the spinal cord, and in free nerve endings in the skin.  To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse.  This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder.  However, DRG and spinal cord tissues from Entpd3-/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates.  Additionally, using fast-scan cyclic voltammetry (FSCV, adenosine production was not impaired in the dorsal spinal cord of Entpd3-/- mice when the substrate ADP was applied.  Further, Entpd3-/- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3-/- mice showed a modest reduction in β-alanine-mediated itch.  Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord.  Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.

  18. Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord [v2; ref status: indexed, http://f1000r.es/4dl

    Directory of Open Access Journals (Sweden)

    Eric McCoy

    2014-09-01

    Full Text Available Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides.  Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP to adenosine in primary somatosensory neurons.  Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons.  Ectonucleoside triphosphate diphosphohydrolases (ENTPDs comprise a class of enzymes that dephosphorylate extracellular ATP and ADP.  Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3 was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG, in the dorsal horn of the spinal cord, and in free nerve endings in the skin.  To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse.  This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder.  However, DRG and spinal cord tissues from Entpd3-/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates.  Additionally, using fast-scan cyclic voltammetry (FSCV, adenosine production was not impaired in the dorsal spinal cord of Entpd3-/- mice when the substrate ADP was applied.  Further, Entpd3-/- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3-/- mice showed a modest reduction in β-alanine-mediated itch.  Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord.  Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.

  19. The Role of the CD39/CD73 Purinergic Pathway in Modulating Arterial Thrombosis in Mice

    Science.gov (United States)

    Covarrubias, R; Chepurko, E; Reynolds, A; Huttinger, ZM; Huttinger, R; Stanfill, K; Wheeler, DG; Novitskaya, T; Robson, SC; Dwyer, KM; Cowan, PJ; Gumina, RJ

    2016-01-01

    Objective Circulating blood cells and endothelial cells express Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5’-nucleotidase (CD73). CD39 hydrolyzes extracellular ATP or ADP to AMP. CD73 hydrolyzes AMP to adenosine. The goal of this study was to examine the interplay between CD39 and CD73 cascade in arterial thrombosis. Approach and Results To determine how CD73 activity influences in vivo thrombosis, the time to FeCl3-induced arterial thrombosis was measured in CD73-null mice. In response to 5% FeCl3, but not to 10% FeCl3, there was a significant decrease in the time to thrombosis in CD73-null mice compared to wild-type (WT) mice. In mice overexpressing CD39, ablation of CD73 did not inhibit the prolongation in the time to thrombosis conveyed by CD39 overexpression. However, the CD73 inhibitor α-β-methylene-ADP nullified the prolongation in the time to thrombosis in hC39-Tg/CD73-null mice. To determine if hematopoietic-derived cells or endothelial cell CD39 activity regulates in vivo arterial thrombus, bone marrow transplant studies were conducted. FeCl3-induced arterial thrombosis in chimeric mice revealed a significant prolongation in the time to thrombosis in hCD39-Tg reconstituted WT mice, but not on WT reconstituted hCD39-Tg mice. Monocyte depletion with clodronate-loaded liposomes normalized the time to thrombosis in hCD39-Tg mice compared to hCD39-Tg mice treated with control liposomes, demonstrating that increased CD39 expression on monocytes protects against thrombosis. Conclusions These data demonstrate that ablation of CD73 minimally effects in vivo thrombosis, but increased CD39 expression on hematopoietic-derived cells, especially monocytes, attenuates in vivo arterial thrombosis. PMID:27417582

  20. 17β-Estradiol-Induced Synaptic Rearrangements Are Accompanied by Altered Ectonucleotidase Activities in Male Rat Hippocampal Synaptosomes.

    Science.gov (United States)

    Mitrović, Nataša; Zarić, Marina; Drakulić, Dunja; Martinović, Jelena; Sévigny, Jean; Stanojlović, Miloš; Nedeljković, Nadežda; Grković, Ivana

    2017-03-01

    17β-Estradiol (E2) rapidly, by binding to membrane estrogen receptors, activates cell signaling cascades which induce formation of new dendritic spines in the hippocampus of males as in females, but the interaction with other metabolic processes, such as extracellular adenine nucleotides metabolism, are currently unknown. Extracellular adenine nucleotides play significant roles, controlling excitatory glutamatergic synapses and development of neural circuits and synaptic plasticity. Their precise regulation in the synaptic cleft is tightly controlled by ecto-nucleoside triphosphate diphosphohydrolase (NTPDase)/ecto-5'-nucleotidase (eN) enzyme chain. Therefore, we sought to clarify whether a single systemic injection of E2 in male rats is accompanied by changes in the expression of the pre- and postsynaptic proteins and downstream kinases linked to E2-induced synaptic rearrangement as well as alterations in NTPDase/eN pathway in the hippocampal synaptosomes. Obtained data showed activation of mammalian target of rapamycin and upregulation of key synaptic proteins necessary for spine formation, 24 h after systemic E2 administration. In E2-mediated conditions, we found downregulation of NTPDase1 and NTPDase2 and attenuation of adenine nucleotide hydrolysis by NTPDase/eN enzyme chain, without changes in NTPDase3 properties and augmentation of synaptic tissue-nonspecific alkaline phosphatase (TNAP) activity. Despite reduced NTPDase activities, increased TNAP activity probably prevents toxic accumulation of ATP in the extracellular milieu and also hydrolyzes accumulated ADP due to unchanged NTPDase3 activity. Thus, our initial evaluation supports idea of specific roles of different ectonucleotidases and their coordinated actions in E2-mediated spine remodeling and maintenance.

  1. Contribution of adenosine-producing ectoenzymes to the mechanisms underlying the mitigation of maternal-fetal conflicts.

    Science.gov (United States)

    Cecati, M; Emanuelli, M; Giannubilo, S R; Quarona, V; Senetta, R; Malavasi, F; Tranquilli, A L; Saccucci, F

    2013-01-01

    The interactions taking place between mother and embryo have been the focus of detailed studies in recent years, where pregnancy is considered as an in vivo transplant. The immune systems of the mother and the embryo together establish a condition of tolerance, which lasts throughout the pregnancy. Alongside immunogenetic components, a contribution is provided by the ectoenzyme network, a chain of surface molecules mainly operating in closed environments and potentially providing inhibitory or activator signals. One of the soluble products of the ectoenzyme network with immunosuppressory potential is adenosine, a purine nucleoside that plays multiple roles in almost all tissues and organs. The hypothesis behind the work was studied in patients with recurrent pregnancy loss (RPL), an event which remains unexplained in over 50 percent of cases. To this aim, we analyzed the expression of CD39 (ectonucleoside triphosphate diphosphohydrolase 1, ENTPD1) and CD73 (ecto-5’-nucleotidase, NT5E), the main pathway for adenosine generation, in samples obtained from women with RPL. The study included the evaluation of the expression of TNF-alpha (a pro-inflammatory cytokine) and of an alternative pathway of adenosine generation run by CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) and PC-1 (ectonucleotide pyrophosphatase/phosphodiesterase 1, ENPP1). The results of this study highlight the existence of a network of surface enzymes expressed at the maternal/fetal interface and addressed to the production of adenosine. Perturbation of this network may induce a rescue pathway driven by CD38 and ENPP1. Ectoenzyme and inflammation may be considered now key elements in orchestrating the events leading to the interruption of pregnancy in the RPL sample analyzed and at the same potentially becoming therapeutic targets.

  2. Acidosis is a key regulator of osteoblast ecto-nucleotidase pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity.

    Science.gov (United States)

    Orriss, Isabel R; Key, Michelle L; Hajjawi, Mark O R; Millán, José L; Arnett, Timothy R

    2015-12-01

    Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi ) to pyrophosphate (PPi ) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi , a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto-nucleotidases. This study investigated the expression and activity of ecto-nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto-nucleotidases including NTPdase 1-6 (ecto-nucleoside triphosphate diphosphohydrolase) and NPP1-3 (ecto-nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 >  alkaline phosphatase > ecto-5-nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8-fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto-nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (P key substrates for NPP1, from osteoblasts, was unaffected by acidosis. Further studies showed that mineralised bone formation by osteoblasts cultured from NPP1 knockout mice was increased compared with wildtypes (2.5-fold, P < 0.001) and was partially resistant to the inhibitory effect of acidosis. These results indicate that increased NPP1 expression and activity might contribute to the decreased mineralisation observed when osteoblasts are exposed to acid conditions. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

  3. Synthesis and evaluation of 2-ethynyl-adenosine-5'-triphosphate as a chemical reporter for protein AMPylation.

    Science.gov (United States)

    Creech, Christa; Kanaujia, Mukul; Causey, Corey P

    2015-08-21

    Protein AMPylation is a posttranslational modification (PTM) defined as the transfer of an adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl side-chain of a protein substrate. One recently reported AMPylator enzyme, Vibrio outer protein S (VopS), plays a role in pathogenesis by AMPylation of Rho GTPases, which disrupts crucial signaling pathways, leading to eventual cell death. Given the resurgent interest in this modification, there is a critical need for chemical tools that better facilitate the study of AMPylation and the enzymes responsible for this modification. Herein we report the synthesis of 2-ethynyl-adenosine-5'-triphosphate () and its utilization as a non-radioactive chemical reporter for protein AMPylation.

  4. Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids.

    Science.gov (United States)

    Hocek, Michal; Fojta, Miroslav

    2008-07-07

    Construction of functionalized nucleic acids (DNA or RNA) via polymerase incorporation of modified nucleoside triphosphates is reviewed and selected applications of the modified nucleic acids are highlighted. The classical multistep approach for the synthesis of modified NTPs by triphosphorylation of modified nucleosides is compared to the novel approach consisting of direct aqueous cross-coupling reactions of unprotected halogenated nucleoside triphosphates. The combination of cross-coupling of NTPs with polymerase incorporation gives an efficient and straightforward two-step synthesis of modified nucleic acids. Primer extension using biotinylated templates followed by separation using streptavidine-coated magnetic beads and DNA duplex denaturation is used for preparation of modified single stranded oligonucleotides. Examples of using this approach for electrochemical DNA labelling and bioanalytical applications are given.

  5. Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans.

    Science.gov (United States)

    Blundell, Ross D; Williams, Simon J; Arras, Samantha D M; Chitty, Jessica L; Blake, Kirsten L; Ericsson, Daniel J; Tibrewal, Nidhi; Rohr, Jurgen; Koh, Y Q Andre E; Kappler, Ulrike; Robertson, Avril A B; Butler, Mark S; Cooper, Matthew A; Kobe, Bostjan; Fraser, James A

    2016-09-09

    Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.

  6. Hybrid integrated biological-solid-state system powered with adenosine triphosphate

    Science.gov (United States)

    Roseman, Jared M.; Lin, Jianxun; Ramakrishnan, Siddharth; Rosenstein, Jacob K.; Shepard, Kenneth L.

    2015-12-01

    There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na+/K+ adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 106 mm-2) are able to sustain a short-circuit current of 32.6 pA mm-2 and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm-2 from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

  7. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration

    Science.gov (United States)

    Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-01-01

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca2+ signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca2+-dependent kinases and phosphatases. Ca2+ channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca2+ homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies. PMID:27658230

  8. In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver

    Directory of Open Access Journals (Sweden)

    Ana V. Frontini

    2011-04-01

    Full Text Available The utilization of adenosine 5´-triphosphate (ATP infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.

  9. The role of adenosine triphosphate in the function of human origin recognition complex 4 protein

    Directory of Open Access Journals (Sweden)

    ALEKSANDRA DIVAC

    2010-03-01

    Full Text Available Human origin recognition complex 4 (ORC4 protein, a subunit of the origin recognition complex, belongs to the AAA+ superfamily of adenosine triphosphate (ATP ases. Proteins belonging to this family require ATP for their function and interactions with ATP lead to conformational changes in them or in their partners. Human ORC4 protein induces structural changes in DNA substrates, promoting renaturation and formation of non-canonical structures, as well as conversion of single-stranded into multi-stranded oligonucleotide structures. The aim of this study was to further investigate the role of ATP in the function of human ORC4 protein. For this purpose, a mutant in the conserved Walker B motif of ORC4, which is able to bind but not to hydrolyze ATP, was constructed and its activity in DNA restructuring reactions was investigated. The obtained results showed that ATP hydrolysis is not necessary for the function of human ORC4. It is proposed that ATP has a structural role as a cofactor in the function of human ORC4 as a DNA restructuring agent.

  10. A novel conductometric biosensor based on hexokinase for determination of adenosine triphosphate.

    Science.gov (United States)

    Kucherenko, I S; Kucherenko, D Yu; Soldatkin, O O; Lagarde, F; Dzyadevych, S V; Soldatkin, A P

    2016-04-01

    The paper presents a simple and inexpensive reusable biosensor for determination of the concentration of adenosine-5'-triphosphate (ATP) in aqueous samples. The biosensor is based on a conductometric transducer which contains two pairs of gold interdigitated electrodes. An enzyme hexokinase was immobilized onto one pair of electrodes, and bovine serum albumin-onto another pair (thus, a differential mode of measurement was used). Conditions of hexokinase immobilization on the transducer by cross-linking via glutaraldehyde were optimized. Influence of experimental conditions (concentration of magnesium ions, ionic strength and concentration of the working buffer) on the biosensor work was studied. The reproducibility of biosensor responses and operational stability of the biosensor were checked during one week. Dry storage at -18 °C was shown to be the best conditions to store the biosensor. The biosensor was successfully applied for measurements of ATP concentration in pharmaceutical samples. The proposed biosensor may be used in future for determination of ATP and/or glucose in water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection.

    Science.gov (United States)

    Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia

    2015-01-01

    A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

  12. Intracellular and extracellular adenosine triphosphate in regulation of insulin secretion from pancreatic β cells (β).

    Science.gov (United States)

    Wang, Chunjiong; Geng, Bin; Cui, Qinghua; Guan, Youfei; Yang, Jichun

    2014-03-01

    Adenosine triphosphate (ATP) synthesis and release in mitochondria play critical roles in regulating insulin secretion in pancreatic β cells. Mitochondrial dysfunction is mainly characterized by a decrease in ATP production, which is a central event in the progression of pancreatic β cell dysfunction and diabetes. ATP has been demonstrated to regulate insulin secretion via several pathways: (i) Intracellular ATP directly closes ATP-sensitive potassium channel to open L-type calcium channel, leading to an increase in free cytosolic calcium levels and exocytosis of insulin granules; (ii) A decrease in ATP production is always associated with an increase in production of reactive oxygen species, which exerts deleterious effects on pancreatic β cell survival and insulin secretion; and (iii) ATP can be co-secreted with insulin from pancreatic β cells, and the released ATP functions as an autocrine signal to modulate insulin secretory process via P2 receptors on the cell membrane. In this review, the recent findings regarding the role and mechanism of ATP synthesis and release in regulation of insulin secretion from pancreatic β cells will be summarized and discussed. © 2013 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  13. Effects of Adenosine Triphosphate on Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells.

    Science.gov (United States)

    Wang, Wei; Yi, Xiaosong; Ren, Yanfang; Xie, Qiufei

    2016-10-01

    Adenosine 5'-triphosphate (ATP) is a potent signaling molecule that regulates diverse biological activities in cells. Its effects on human dental pulp cells (HDPCs) remain unknown. This study aimed to examine the effects of ATP on proliferation and differentiation of HDPCs. Reverse transcription polymerase chain reaction was performed to explore the mRNA expression of P2 receptor subtypes. Cell Counting Kit-8 test and flow cytometry analysis were used to examine the effects of ATP on proliferation and cell cycle of HDPCs. The effects of ATP on differentiation of HDPCs were examined by using alizarin red S staining, energy-dispersive x-ray analysis, Western blot analysis, and real-time polymerase chain reaction. The purinoceptors P2X3, P2X4, P2X5, P2X7, and all P2Y receptor subtypes were confirmed to present in HDPCs. ATP enhanced HDPC proliferation at 10 μmol/L concentration. However, it inhibited cell proliferation by arresting the cell cycle in G0G1 phase (P concentration. Suramin, an ATP receptor antagonist, inhibited ERK/MAPK activation and HDPC odontoblastic differentiation (P dental pulp tissue healing and repair through P2 signaling. Results provide new insights into the molecular regulation of pulpal wound healing. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  14. HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway

    Directory of Open Access Journals (Sweden)

    Ana White

    2012-02-01

    The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP3levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP3 signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP3 levels during the reproductive cycle.

  15. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis.

    Science.gov (United States)

    Jastrab, Jordan B; Wang, Tong; Murphy, J Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P; Li, Huilin; Darwin, K Heran

    2015-04-07

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.

  16. Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

    Science.gov (United States)

    Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

    1977-01-01

    The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

  17. Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Simonova, Anna; Bialek-Pietras, M.; Olejniczak, A.; Lesnikowski, Z. J.; Hocek, Michal

    2017-01-01

    Roč. 27, č. 21 (2017), s. 4786-4788 ISSN 0960-894X R&D Projects: GA ČR GBP206/12/G151 Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : nucleotides * nucleoside triphosphates * carboranes * DNA polymerase * oligonucleotides Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.454, year: 2016

  18. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

    Science.gov (United States)

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

    Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

  19. Dual hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by Orf17 (NtpA), a MutT-type enzyme.

    Science.gov (United States)

    Hori, Mika; Fujikawa, Katsuyoshi; Kasai, Hiroshi; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2005-01-02

    To determine whether the Orf17 (NtpA) protein of Escherichia coli, a MutT-type enzyme, functions as a hydrolyzing enzyme for a damaged deoxyribonucleotide, we purified the recombinant Orf17 protein and incubated it with oxidized deoxyribonucleotides. Of the deoxyribonucleoside 5'-triphosphates tested, 8-hydroxy-2'-deoxyadenosine 5'-triphosphate was hydrolyzed by this protein. Unexpectedly, the Orf17 protein degraded 8-hydroxy-2'-deoxyadenosine 5'-diphosphate 2.3-fold more efficiently than the corresponding triphosphate. Thus, this protein is the first MutT-type enzyme that hydrolyzes both the triphosphate and diphosphate derivatives of a deoxyribonucleoside, with similar efficiencies. These results suggest that the Orf17 protein may be involved in the hydrolysis of oxidized dATP and dADP.

  20. Pivotal role of inosine triphosphate pyrophosphatase in maintaining genome stability and the prevention of apoptosis in human cells.

    Science.gov (United States)

    Menezes, Miriam Rose; Waisertreiger, Irina S-R; Lopez-Bertoni, Hernando; Luo, Xu; Pavlov, Youri I

    2012-01-01

    Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.

  1. HILIC LC-MS for the determination of 2'-C-methyl-cytidine-triphosphate in rat liver.

    Science.gov (United States)

    Pucci, Vincenzo; Giuliano, Claudio; Zhang, Rena; Koeplinger, Kenneth Alan; Leone, Joseph F; Monteagudo, Edith; Bonelli, Fabio

    2009-05-01

    A very accurate and selective LC-MS/MS method was developed and validated for the quantification of 2'-C-modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96-well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm x 2.0 mm, 3 microm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 microL/min flow rate. The 2'-C-modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05-50 microM concentration range. Satisfying results, confirming the high reliability of the established LC-MS/MS method, were obtained for intraday precision (CV = 2.5-9.1%) and accuracy (92.6-94.8%) and interday precision (CV = 9.6-11.5%) and accuracy (94.4-102.4%) as well as for recovery (82.0-112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2'-C-methyl-cytidine-triphosphate in liver tissue samples.

  2. Ternary Interactions and Energy Transfer between Fluorescein Isothiocyanate, Adenosine Triphosphate, and Graphene Oxide Nanocarriers.

    Science.gov (United States)

    Ratajczak, Katarzyna; Stobiecka, Magdalena

    2017-07-20

    The interactions of fluorescent probes and biomolecules with nanocarriers are of key importance to the emerging targeted drug delivery systems. Graphene oxide nanosheets (GONs) as the nanocarriers offer biocompatibility and robust drug binding capacity. The interactions of GONs with fluorophores lead to strong fluorescence quenching, which may interfere with fluorescence bioimaging and biodetection. Herein, we report on the interactions and energy transfers in a model ternary system: GONs-FITC-ATP, where FITC is a model fluorophore (fluorescein isothiocyanate) and ATP is a common biomolecule (adenosine-5'-triphosphate). We have found that FITC fluorescence is considerably quenched by ATP (the quenching constant K SV = 113 ± 22 M -1 ). The temperature coefficient of K SV is positive (α T = 4.15 M -1 deg -1 ). The detailed analysis of a model for internal self-quenching of FITC indicates that the temperature dependence of the net quenching efficiency η for the FITC-ATP pair is dominated by FITC internal self-quenching modes with their contribution estimated at 79%. The quenching of FITC by GONs is much stronger (K SV = 598 ± 29 M -1 ) than that of FITC-ATP and is associated with the formation of supramolecular assemblies bound with hydrogen bonding and π-π stacking interactions. For the analysis of the complex behavior of the ternary system GONs-FITC-ATP, a model of chemisorption of ATP on GONs, with partial blocking of FITC quenching, has been developed. Our results indicate that ATP acts as a moderator for FITC quenching by GONs. The interactions between ATP, FITC, and GONs have been corroborated using molecular dynamics and quantum mechanical calculations.

  3. Deoxyribonucleoside triphosphate pools and thymidine chemosensitization in human T-cell leukemia.

    Science.gov (United States)

    Cohen, J D; Robins, H I; Katz, T B; Miller, E M; Kuzminsky, S R; Javid, M J

    1993-02-01

    Thymidine kills cells by depleting dCTP stores. The present experiments tested whether deoxycytidine, by replenishing dCTP pools, could prevent thymidine cytotoxicity and thymidine's enhancement of carboplatin killing in two human T-cell acute leukemia cell lines. MOLT3 and JM cells were exposed to combinations of thymidine, deoxycytidine, and carboplatin and then assessed for survival, the magnitude of thymidine-carboplatin chemosensitization, and changes in deoxyribonucleoside triphosphate pools. For both cell lines, deoxycytidine (up to 144.5 micrograms/ml x 24 h) completely restored dCTP pools but only partially protected against thymidine cytotoxicity (100-1000 micrograms/ml x 24 h) and thymidine-carboplatin sensitization (up to 60 micrograms carboplatin/ml during the last hour of thymidine). This contrasts with complete protection in prior studies using other cell types. Thymidine alone markedly increased dTTP and dGTP pools and decreased dCTP; dATP pools underwent a sharp decline which has not been observed before in any cell line. In subsequent studies 0.0336-137.3 micrograms deoxyadenosine/ml partially prevented cytotoxicity and carboplatin sensitization by 300 micrograms thymidine/ml. Together, deoxycytidine and deoxyadenosine completely prevented thymidine-carboplatin sensitization even though dATP and dCTP pools were not entirely returned to normal. These findings are discussed in regard to the unusual sensitivity of T-cell malignancies to thymidine toxicity, mechanisms of cytotoxicity and chemosensitization by thymidine, and the possibility of thymidine selectively sensitizing T-cell malignancies to killing by alkylating agents.

  4. Potentiation of Muscarinic and α -adrenergic Responses by an Analogue of Guanosine 5'-triphosphate

    Science.gov (United States)

    Evans, M. G.; Marty, A.

    1986-06-01

    Ca2+-dependent K+ and Cl- currents were recorded in isolated and dialyzed rat lacrimal gland cells by use of the tight-seal whole-cell recording technique. Under control conditions, application of acetylcholine (0.5-1.0 μ M) resulted in the full activation of both types of current. When 50-200 μ M guanosine 5'-[γ -thio]triphosphate (GTP[S], a nonhydrolyzable GTP analogue) was added to the intracellular solution, activation of both currents was seen with 1 nM acetylcholine, a dose 1/100th that needed under control conditions. Dialysis with solutions containing 200 μ M GTP or cAMP had no, or only slight, potentiation effects. The effects of GTP[S] were obtained only when ATP was included in the intracellular solution. The potentiated responses to acetylcholine were blocked by increasing 10-fold the intracellular Ca2+-buffering capacity and were not dependent on external Ca2+. Thus, the potentiated responses appeared to result from a release of Ca2+ from internal stores. GTP[S] also greatly potentiated the Ca2+-dependent adrenergic (norepinephrine) response of this preparation. In addition, GTP[S] elicited in some cells transient responses without application of acetylcholine or norepinephrine. Finally, rapid and sustained responses were seen as soon as the cells were dialyzed with inositol trisphosphate (20 μ M). These findings are discussed in terms of a possible role of a GTP-binding protein as a link between activation of muscarinic or adrenergic receptors and initiation of Ca2+ release by inositol trisphosphate.

  5. Beneficial effect of propofol on arterial adenosine triphosphate-sensitive K+ channel function impaired by thromboxane.

    Science.gov (United States)

    Haba, Masanori; Kinoshita, Hiroyuki; Matsuda, Naoyuki; Azma, Toshiharu; Hama-Tomioka, Keiko; Hatakeyama, Noboru; Yamazaki, Mitsuaki; Hatano, Yoshio

    2009-08-01

    It is not known whether thromboxane A2 impairs adenosine triphosphate (ATP)-sensitive K channel function via increased production of superoxide in blood vessels and whether propofol as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor restores this modification. Rat aortas without endothelium were used for isometric force recording, measurements of membrane potential, and superoxide production and Western immunoblotting. Vasorelaxation to an ATP-sensitive K channel opener levcromakalim was obtained during contraction to phenylephrine (3 x 10(-7) M) or a thromboxane A2 analogue U46619 (3 x 10(-7) M). In some experiments, aortas were incubated with an ATP-sensitive K channel antagonist glibenclamide, a superoxide inhibitor Tiron, a nonselective NADPH oxidase inhibitor apocynin, a hydrogen peroxide scavenger catalase, a xanthine oxidase inhibitor allopurinol, a thromboxane receptor antagonist SQ29548 or propofol (3 x 10(-7) to 3 x 10(-6) M). Levcromakalim-induced vasorelaxation was abolished by glibenclamide in rings contracted with either vasoconstrictor agent. Tiron, apocynin, and propofol, but not catalase, augmented the vasodilator response as well as the hyperpolarization by levcromakalim in aortas contracted with U46619. Tiron, apocynin, SQ29548, and propofol, but not allopurinol, similarly reduced in situ levels of superoxide within aortic vascular smooth muscle exposed to U46619. Protein expression of a NADPH oxidase subunit p47phox increased in these arteries, and this augmentation was abolished by propofol. Thromboxane receptor activation induces vascular oxidative stress via NADPH oxidase, resulting in the impairment of ATP-sensitive K channel function. Propofol reduces this stress via inhibition of a NADPH oxidase subunit p47phox and, therefore, restores ATP-sensitive K channel function.

  6. Pharmacokinetic evaluation of the effects of ribavirin on zidovudine triphosphate formation: ACTG 5092s Study Team.

    Science.gov (United States)

    Aweeka, F T; Kang, M; Yu, J-Y; Lizak, P; Alston, B; Chung, R T

    2007-07-01

    Ribavirin (RBV) is used for the treatment of hepatitis C virus (HCV) infection in subjects with HIV-1 infection who may require antiretroviral treatment (ART) with nucleoside reverse transcriptase inhibitors including zidovudine (ZDV). We sought to investigate the potential antagonism between RBV and ZDV by evaluating the impact of RBV on the formation of intracellular ZDV triphosphate (TP) in HIV-infected patients receiving ZDV who were treated for HCV infection. Serial plasma and intracellular ZDV TP pharmacokinetics (PK) were determined in 14 subjects at entry (within 2 weeks prior to RBV administration) and at 8 weeks following initiation of RBV. Intracellular ZDV TP in peripheral blood mononuclear cells (PBMC) was quantified by a validated cartridge/liquid chromatography/tandem mass spectrometry method. PK exposure was estimated from the steady-state area under the concentration vs time curve (AUC(0-12 h)) in plasma and PBMC. Ribavirin did not have a statistically significant impact on ZDV TP AUC(0-12 h), plasma ZDV AUC(0-12 h) or the ratio of ZDV TP AUC(0-12 h) to plasma ZDV AUC(0-12 h), although there was a trend towards an increase post-RBV ratio compared with pre-RBV. There was extensive variability in the ZDV TP AUC(0-12 h). Ribavirin did not inhibit formation of ZDV TP in PBMC in 14 patients receiving ZDV as part of ART and RBV-based HCV therapy for 8 weeks. These results are consistent with those of a previously published limited study in seven subjects. These PK findings should be weighed carefully against emerging clinical reports of significant anaemia associated with combination ZDV and high-dose RBV therapy.

  7. Adenosine 5'-Triphosphate Test in the Management of Patients With Syncope.

    Science.gov (United States)

    Flammang, Daniel; Benditt, David G; Church, Timothy R; Pelleg, Amir

    The response to adenosine 5'-triphosphate (ATP) identifies patients with syncope who might benefit from pacemaker therapy (ATP test). Two measures have been used to determine the outcome of the ATP test, which have lead to contrasting conclusions regarding its utility: (1) the duration of cardiac pause (CP) mainly due to AV block and (2) the longest RR interval (RRmax). We tested the hypothesis that the discrepancy regarding the utility of the ATP test is mainly because of the different way the 2 measures determine the outcome of the test. Post hoc analysis was applied to data obtained from patients with syncope (n = 33) with a positive and negative ATP test based on the CP duration and RRmax, respectively, subjected to pacemaker therapy. In 19 and 14 patients, the pacemaker was programmed to function as AAI pacing at 30 ppm (control) and as DDD pacing at 70 ppm, respectively. During the follow-up period of 17.0 ± 8.6 months, syncope recurred in only 1 of the 14 patients with DDD pacing; in contrast, 10 of 19 patients with AAI30 pacing experienced syncope within the first 5.3 ± 5.2 months of follow-up (P syncope who may benefit from pacemaker therapy; the identification of such patients would be missed when the RRmax measure is used to determine the outcome of the test. The efficacy of DDD pacing suggests that atrioventricular nodal conduction block is the primary cause of syncope in patients with a positive ATP test based on the CP measure.

  8. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5⿲ triphosphate (ATP)

    Science.gov (United States)

    Hammami, Khaled; El-Feki, Hafed; Marsan, Olivier; Drouet, Christophe

    2016-01-01

    ATP is a well-known energy supplier in cells. The idea to associate ATP to pharmaceutical formulations/biotechnological devices to promote cells activity by potentially modulating their microenvironment thus appears as an appealing novel approach. Since biomimetic nanocrystalline apatites have shown great promise for biomedical applications (bone regeneration, cells diagnostics/therapeutics, ⿦), thanks to a high surface reactivity and an intrinsically high biocompatibility, the present contribution was aimed at exploring ATP/apatite interactions. ATP adsorption on a synthetic carbonated nanocrystalline apatite preliminarily characterized (by XRD, FTIR, Raman, TG-DTA and SEM-EDX) was investigated in detail, pointing out a good agreement with Sips isothermal features. Adsorption characteristics were compared to those previously obtained on monophosphate nucleotides (AMP, CMP), unveiling some specificities. ATP was found to adsorb effectively onto biomimetic apatite: despite smaller values of the affinity constant KS and the exponential factor m, larger adsorbed amounts were reached for ATP as compared to AMP for any given concentration in solution. m guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard οGads ° was estimated to only ⿿4 kJ/mol, the large value of Nmax led to significantly negative effective οGads values down to ⿿33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, ⿦).

  9. Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription.

    Science.gov (United States)

    Gross, C H; Shuman, S

    1996-12-01

    Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.

  10. Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation.

    Directory of Open Access Journals (Sweden)

    Qian Feng

    Full Text Available Upon viral infections, pattern recognition receptors (PRRs recognize pathogen-associated molecular patterns (PAMPs and stimulate an antiviral state associated with the production of type I interferons (IFNs and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5' triphosphate (5'ppp-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3, a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5'ppp. We show here that a 5'ppp-containing cloverleaf (CL RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.

  11. CaMKII Regulation of Cardiac Ryanodine Receptors and Inositol Triphosphate Receptors

    Directory of Open Access Journals (Sweden)

    Emmanuel eCamors

    2014-05-01

    Full Text Available Ryanodine receptors (RyRs and inositol triphosphate receptors (InsP3Rs are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR, and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and posttranslational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

  12. Molecular structure of tetraaqua adenosine 5'-triphosphate aluminium(III) complex: A study involving Raman spectroscopy, theoretical DFT and potentiometry

    Science.gov (United States)

    Tenório, Thaís; Silva, Andréa M.; Ramos, Joanna Maria; Buarque, Camilla D.; Felcman, Judith

    2013-03-01

    The Alzheimer's disease is one of the most common neurodegenerative diseases that affect elderly population, due to the formation of β-amyloid protein aggregate and several symptoms, especially progressive cognitive decline. The result is a decrease in capture of glucose by cells leading to obliteration, meddling in the Krebs cycle, the principal biochemical route to the energy production leading to a decline in the levels of adenosine 5'-triphosphate. Aluminium(III) is connected to Alzheimer's and its ion provides raise fluidity of the plasma membrane, decrease cell viability and aggregation of amyloid plaques. Studies reveal that AlATP complex promotes the formation of reactive fibrils of β-amyloid protein and independent amyloidogenic peptides, suggesting the action of the complex as a chaperone in the role pathogenic process. In this research, one of complexes formed by Al(III) and adenosine 5'-triphosphate in aqueous solution is analyzed by potentiometry, Raman spectroscopy and ab initio calculations. The value of the log KAlATP found was 9.21 ± 0.01 and adenosine 5'-triphosphate should act as a bidentate ligand in the complex. Raman spectroscopy and potentiometry indicate that donor atoms are the oxygen of the phosphate β and the oxygen of the phosphate γ, the terminal phosphates. Computational calculations using Density Functional Theory, with hybrid functions B3LYP and 6-311++G(d,p) basis set regarding water solvent effects, have confirmed the results. Frontier molecular orbitals, electrostatic potential contour surface, electrostatic potential mapped and Mulliken charges of the title molecule were also investigated.

  13. Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

    Directory of Open Access Journals (Sweden)

    Sébastien Plumet

    Full Text Available BACKGROUND: Double stranded RNA (dsRNA is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader.

  14. Nitrogen and phosphorus co-doped graphene quantum dots: synthesis from adenosine triphosphate, optical properties, and cellular imaging.

    Science.gov (United States)

    Ananthanarayanan, Arundithi; Wang, Yue; Routh, Parimal; Sk, Mahasin Alam; Than, Aung; Lin, Ming; Zhang, Jie; Chen, Jie; Sun, Handong; Chen, Peng

    2015-05-07

    Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of applications, particularly, as superior fluorescent reporters for bio-imaging and optical sensing. Heteroatom doping can endow GQDs with new or improved photoluminescence properties. Here, we demonstrate a simple strategy for the synthesis of nitrogen and phosphorus co-doped GQDs from a single biomolecule precursor (adenosine triphosphate - ATP). Such ATP-GQDs exhibit high fluorescence quantum yield, strong two-photon upconversion, small molecular weight, high photostability, and good biocompatibility. Furthermore, transferrin conjugated ATP-GQDs have been used for imaging and real-time tracking of transferrin receptors in live cells.

  15. Adenosine 5'-triphosphate formation in Thiobacillus ferrooxidans vesicles by H+ ion gradients comparable to those of environmental conditions.

    OpenAIRE

    Apel, W A; Dugan, P R; Tuttle, J H

    1980-01-01

    Vesicles prepared from iron-grown Thiobacillus ferrooxidans, and subsequently loaded with adenosine 5'-diphosphate and inorganic phosphate, produced adenosine 5'-triphosphate when subjected to H+ gradients comparable to those in the cells' normal environment (i.e., an internal pH in the range of 6.0 to 8.0 with an optimum of 7.0 to 7.8 and an external pH in the range of 2.1 to 4.1 with an optimum of 2.8). Nigericin, dicyclohexylcarbodiimide, and pentachlorophenol decreased adenosine 5'-tripho...

  16. Adenosine triphosphate levels during anaphylactic histamine release in rat mast cells in vitro. Effects of glycolytic and respiratory inhibitors

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The adenosine triphosphate (ATP) content of rat mast cells was studied during and after anaphylactic histamine release. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors supports the view that the ATP depletion was largely...... related to the histamine release process and not to an uncoupling of oxidative phosphorylation by an increased concentration of cytosol Ca2+. The ATP content of the cells was not restored within the 2 h of observation. No inhibition of lactate production from mast cells exposed to antigen in the presence...

  17. SUCCESSFUL TREATMENT OF PAROXYSMAL SUPRAVENTRICULAR TACHYCARDIA IN A LOW-BIRTH-WEIGHT NEWBORN BY INJECTION OF ADENOSINE-5'-TRIPHOSPHATE

    OpenAIRE

    古市, 康子; 吉田, 裕慈; 川口, 千晴; 桑原, 勲; 箕輪, 秀樹; 江國, 豊; 吉岡, 章; 高橋, 幸博; 松村, 正彦

    2002-01-01

    We report a case in which paroxysmal supraventricular tachycardia (PSVT) in a low-birth-weight newborn, due to atrioventricular nodal re-entry, wassuccessfully treated by intravenous bolus injection of adenosine-5'-triphosphate (ATP). The newborn was a male, born after 36 weeks and 1 day of gestation, weighing 2,204g. One hour after birth, he exhibited tachycardia, at 250 to 270 beats/min. A chest radiogragh revealed cardiomegaly, with a cardiothoracic ratio of 71%. Injection of ATP at a dose...

  18. Rosuvastatin lowers coenzyme Q10 levels, but not mitochondrial adenosine triphosphate synthesis, in children with familial hypercholesterolemia.

    Science.gov (United States)

    Avis, Hans J; Hargreaves, Ian P; Ruiter, Jos P N; Land, John M; Wanders, Ronald J; Wijburg, Frits A

    2011-03-01

    To investigate whether statin therapy affects coenzyme Q10 (CoQ10) status in children with heterozygous familial hypercholesterolemia (FH). Samples were obtained at baseline (treatment naïve) and after dose titration with rosuvastatin, aiming for a low-density lipoprotein cholesterol level of 110 mg/dL. Twenty-nine patients were treated with 5, 10, or 20 mg of rosuvastatin for a mean period of 29 weeks. We found a significant (32%) decrease in peripheral blood mononuclear cell (PBMC) CoQ10 level (P = .02), but no change in PBMC adenosine triphosphate synthesis (P = .60). Uncorrected plasma CoQ10 values were decreased significantly, by 45% (P treatment. In children with FH, rosuvastatin causes a significant decrease in cellular PBMC CoQ10 status but does not affect mitochondrial adenosine triphosphate synthesis in children with FH. Further studies should address whether (rare) side effects of statin therapy could be explained by a deterioration in CoQ10 status. Copyright © 2011 Mosby, Inc. All rights reserved.

  19. Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction.

    Science.gov (United States)

    Xia, Zuyong; Azurmendi, Hugo F; Mildvan, Albert S

    2005-11-22

    The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate

  20. Adenosine Triphosphate-Triggered Release of Macromolecular and Nanoparticle Loads from Aptamer/DNA-Cross-Linked Microcapsules.

    Science.gov (United States)

    Liao, Wei-Ching; Lu, Chun-Hua; Hartmann, Raimo; Wang, Fuan; Sohn, Yang Sung; Parak, Wolfgang J; Willner, Itamar

    2015-09-22

    The synthesis of stimuli-responsive DNA microcapsules acting as carriers for different payloads, and being dissociated through the formation of aptamer-ligand complexes is described. Specifically, stimuli-responsive anti-adenosine triphosphate (ATP) aptamer-cross-linked DNA-stabilized microcapsules loaded with tetramethylrhodamine-modified dextran (TMR-D), CdSe/ZnS quantum dots (QDs), or microperoxidase-11 (MP-11) are presented. In the presence of ATP as trigger, the microcapsules are dissociated through the formation of aptamer-ATP complexes, resulting in the release of the respective loads. Selective unlocking of the capsules is demonstrated, and CTP, GTP, or TTP do not unlock the pores. The ATP-triggered release of MP-11 from the microcapsules enables the MP-11-catalyzed oxidation of Amplex UltraRed by H2O2 to the fluorescent product resorufin.

  1. A rapid method for the determination of microbial susceptibility using the firefly luciferase assay for adenosine triphosphate (ATP)

    Science.gov (United States)

    Vellend, H.; Tuttle, S. A.; Barza, M.; Weinstein, L.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was optimized for pure bacteria in broth in order to evaluate if changes in bacterial ATP content could be used as a rapid measure of antibiotic effect on microorganisms. Broth cultures of log phase bacteria were incubated at 310 K (37 C) for 2.5 hours at antimicrobial concentrations which resulted in the best discrimination between sensitive and resistant strains. Eighty-seven strains of 11 bacterial species were studied for their susceptibility to 12 commonly used antimicrobial agents: ampicillin, Penicillin G, nafcillin, carbenicillin, cephalothin, tetracycline, erythromycin, clindamycin, gentamicin, nitrofurantoin, colistin, and chloramplenicol. The major advantage of the ATP system over existing methods of rapid microbial susceptibility testing is that the assay can be made specific for bacterial ATP.

  2. Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast...... cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p...... The observations are consistent with the view that energy requiring processes are involved in ionophore-induced histamine release from rat mast cells although part of the ATP reduction in the aerobic experiments may be due to an uncoupling effect of calcium on the oxidative phosphorylation....

  3. An Escherichia coli strain deficient for both exonuclease 5 and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Estevenon, A.M.; Kooistra, J.; Sicard, N.

    1995-01-01

    An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity

  4. Unique energetic properties of Adenosine Tri-Phosphate in comparison to similar compounds using density functional theory

    Science.gov (United States)

    Muraszko, Kevin; Halloran, Thomas; Malinovskaya, Svetlana; Leopold, Philip

    2015-05-01

    Adenosine Tri-Phosphate (ATP) is arguably the most critical compound to all life known on Earth, serving as the main energy transport and storage in cellular biology. Why in particular did nature ``choose'' ATP instead of a similar compound? We are seeking to answer this question by comparing the energetic properties of ATP to similar compounds. We discuss 3-D models for ATP, variants of the molecule based on all of the separate nucleobases, and ATP's twin molecule Adenosine Di-Phosphate. All calculations were done using Density Functional Theory. The results showed that purine compounds like Adenosine and Guanosine produce similar bond angles, making these viable unlike the other nucleobases. We have analyzed the chiral properties of ATP by comparing the ground-state-energies of ATP-cis and ATP-trans and have shown that ATP-cis is the more energetically favorable of the two. This is consistent with observations in nature.

  5. Isolated cerebellar variant of adrenoleukodystrophy with a de novo adenosine triphosphate-binding cassette D1 (ABCD1) gene mutation.

    Science.gov (United States)

    Kang, Joon Won; Lee, Sang Mi; Koo, Kyo Yeon; Lee, Young-Mock; Nam, Hyo Suk; Quan, Zhejiu; Kang, Hoon-Chul

    2014-07-01

    X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms.

  6. A label-free fluorescent adenosine triphosphate biosensor via overhanging aptamer-triggered enzyme protection and target recycling amplification.

    Science.gov (United States)

    Wang, Zhaoyin; Zhao, Jian; Dai, Zhihui

    2016-06-20

    Herein, a label-free fluorescent adenosine triphosphate (ATP) aptasensor is fabricated with a DNA hairpin and an overhanging aptamer. In the presence of ATP, the overhanging sequences of the aptamer may form preferred substrates of exo III, and thus trigger the enzyme-assisted amplification, which results in the release of G-rich sequences. Free G-rich sequences subsequently generate an enhanced flourescent signal by binding with thioflavin T. However, if ATP is absent, the overhanging sequence can induce steric hindrance and protect the DNA hairpin against the digestion of exo III, significantly reducing the noise of this biosensor. Accordingly, the signal-to-noise ratio of the sensing system is greatly improved, which ensures the desirable analytical performance of the proposed aptasensor both in pure samples and real samples.

  7. Effect molybdenum (5, 6) compounds on hydrolysis and reorganization rates of ammonium and potassium triphosphates in aqueous solutions

    International Nuclear Information System (INIS)

    Petrovskaya, L.I.; Prodan, E.A.; Lesnikovich, L.A.

    1992-01-01

    Method of thin-layer chromatography was used to investigate the kinetics of hydrolytic decomposition of triphosphate-anion in aqueous solutions of M 5 P 3 O 10 -(NH 4 ) 6 xMo 7 O 24 -H 2 O systems (M - NH 4 , pH 5.9-6.4; M - K, pH 6.5-6.8) and (NH 4 ) 5 P 3 O 10 -(NH 4 ) 2 MoOCl 5 -H 2 O system (pH 2.5 and 4.5) at 20 deg C and 0.2 mol% concnetration. It is shown that oxoions Mo 5 produce less accelerating effect on hydrolysis, as compared to Mo 6 oxoions, and are able to initiate anion reorganization with formation of tetraphosphate under certain conditions

  8. Influence of morphology and topography on potentiometric response of magnesium and calcium sensitive PEDOT films doped with adenosine triphosphate (ATP)

    International Nuclear Information System (INIS)

    Paczosa-Bator, B.; Peltonen, J.; Bobacka, J.; Lewenstam, A.

    2006-01-01

    Poly(3,4-ethylenedioxythiophene) (PEDOT) films doped with adenosine triphosphate (ATP) are used to study the biologically relevant competitive magnesium and calcium ion-exchange at ATP membrane sites. It is shown, by atomic force microscopy (AFM) and scanning electron microscopy (SEM), that the surface topography and morphology of the PEDOT-ATP films determines the quality of their potentiometric response. More smooth and less rough films result in better potentiometric characteristics, particularly in a faster response. The topography/morphology of the PEDOT-ATP films is influenced by conditions during electrodeposition (electrochemical method of deposition, pH, concentration of electrolytes) and post-deposition soaking (including net-time of soaking), as evidenced by X-ray photoelectron spectroscopy (XPS) and energy dispersive analysis of X-rays (EDAX)

  9. 7-Aryl-7-deazaadenine 2 '-Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Fanfrlík, Jindřich; Hocek, Michal

    2014-01-01

    Roč. 53, č. 29 (2014), s. 7552-7555 ISSN 1433-7851 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : DNA * enzymes * kinetic s * nucleoside triphosphates * nucleotides Subject RIV: CC - Organic Chemistry Impact factor: 11.261, year: 2014

  10. Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence.

    NARCIS (Netherlands)

    Pellegrini, P.; Sauerwein, R.W.; Finlayson, T.; McLeod, J.; Covell, D.A.; Maier, T.; Machida, C.A.

    2009-01-01

    INTRODUCTION: Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate

  11. Adenosine 5 '-triphosphate (ATP) supplements are not orally bioavailable: a randomized, placebo-controlled cross-over trial in healthy humans

    NARCIS (Netherlands)

    Arts, I.C.W.; Coolen, E.J.C.M.; Bours, M.J.L.; Huyghebaert, N.; Cohen Stuart, M.A.; Bast, A.; Dagnelie, P.C.

    2012-01-01

    Background: Nutritional supplements designed to increase adenosine 5'-triphosphate (ATP) concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity

  12. Synthesis of Base-Modified 2 '-Deoxyribonucleoside Triphosphates and Their Use in Enzymatic Synthesis of Modified DNA for Applications in Bioanalysis and Chemical Biology

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal

    2014-01-01

    Roč. 79, č. 21 (2014), s. 9914-9921 ISSN 0022-3263 R&D Projects: GA ČR GBP206/12/G151; GA ČR GA14-04289S Institutional support: RVO:61388963 Keywords : cross-coupling reactions * modified nucleoside triphosphates * nucleic acids Subject RIV: CC - Organic Chemistry Impact factor: 4.721, year: 2014

  13. An efficient method for the construction of functionalized DNA bearing amino acid groups through cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR

    Czech Academy of Sciences Publication Activity Database

    Čapek, Petr; Cahová, Hana; Pohl, Radek; Hocek, Michal; Gloeckner, Ch.; Marx, A.

    2007-01-01

    Roč. 13, č. 21 (2007), s. 6196-6203 ISSN 0947-6539 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0043 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside triphosphates * cross-coupling * DNA Subject RIV: CC - Organic Chemistry Impact factor: 5.330, year: 2007

  14. Fludarabine-mediated circumvention of cytarabine resistance is associated with fludarabine triphosphate accumulation in cytarabine-resistant leukemic cells.

    Science.gov (United States)

    Yamamoto, Shuji; Yamauchi, Takahiro; Kawai, Yasukazu; Takemura, Haruyuki; Kishi, Shinji; Yoshida, Akira; Urasaki, Yoshimasa; Iwasaki, Hiromichi; Ueda, Takanori

    2007-02-01

    The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5'-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5'-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

  15. Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

    Directory of Open Access Journals (Sweden)

    Shannon E Hill

    Full Text Available Dihydroneopterin triphosphate pyrophosphatase (DHNTPase, a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

  16. Qualitative and quantitative assessment of adenosine triphosphate stress whole-heart dynamic myocardial perfusion imaging using 256-slice computed tomography.

    Science.gov (United States)

    Kurata, Akira; Kawaguchi, Naoto; Kido, Teruhito; Inoue, Katsuji; Suzuki, Jun; Ogimoto, Akiyoshi; Funada, Jun-ichi; Higaki, Jitsuo; Miyagawa, Masao; Vembar, Mani; Mochizuki, Teruhito

    2013-01-01

    The aim of this study was to investigate the correlation of the qualitative transmural extent of hypoperfusion areas (HPA) using stress dynamic whole-heart computed tomography perfusion (CTP) imaging by 256-slice CT with CTP-derived myocardial blood flow (MBF) for the estimation of the severity of coronary artery stenosis. Eleven patients underwent adenosine triphosphate (0.16 mg/kg/min, 5 min) stress dynamic CTP by 256-slice CT (coverage: 8 cm, 0.27 s/rotation), and 9 of the 11 patients underwent coronary angiography (CAG). Stress dynamic CTP (whole-heart datasets over 30 consecutive heart beats in systole without spatial and temporal gaps) was acquired with prospective ECG gating (effective radiation dose: 10.4 mSv). The extent of HPAs was visually graded using a 3-point score (normal, subendocardial, transmural). MBF (ml/100g/min) was measured by deconvolution. Differences in MBF (mean ± standard error) according to HPA and CAG results were evaluated. In 27 regions (3 major coronary territories in 9 patients), 11 coronary stenoses (> 50% reduction in diameter) were observed. In 353 myocardial segments, HPA was significantly related to MBF (P 70%], 119 ± 69). The qualitative transmural extent of HPA using stress whole-heart dynamic CTP imaging by 256-slice CT exhibits a good correlation with quantitative CTP-derived MBF and may aid in assessing the hemodynamic significance of coronary artery disease.

  17. Qualitative and quantitative assessment of adenosine triphosphate stress whole-heart dynamic myocardial perfusion imaging using 256-slice computed tomography.

    Directory of Open Access Journals (Sweden)

    Akira Kurata

    Full Text Available BACKGROUND: The aim of this study was to investigate the correlation of the qualitative transmural extent of hypoperfusion areas (HPA using stress dynamic whole-heart computed tomography perfusion (CTP imaging by 256-slice CT with CTP-derived myocardial blood flow (MBF for the estimation of the severity of coronary artery stenosis. METHODS AND RESULTS: Eleven patients underwent adenosine triphosphate (0.16 mg/kg/min, 5 min stress dynamic CTP by 256-slice CT (coverage: 8 cm, 0.27 s/rotation, and 9 of the 11 patients underwent coronary angiography (CAG. Stress dynamic CTP (whole-heart datasets over 30 consecutive heart beats in systole without spatial and temporal gaps was acquired with prospective ECG gating (effective radiation dose: 10.4 mSv. The extent of HPAs was visually graded using a 3-point score (normal, subendocardial, transmural. MBF (ml/100g/min was measured by deconvolution. Differences in MBF (mean ± standard error according to HPA and CAG results were evaluated. In 27 regions (3 major coronary territories in 9 patients, 11 coronary stenoses (> 50% reduction in diameter were observed. In 353 myocardial segments, HPA was significantly related to MBF (P 70%], 119 ± 69. CONCLUSION: The qualitative transmural extent of HPA using stress whole-heart dynamic CTP imaging by 256-slice CT exhibits a good correlation with quantitative CTP-derived MBF and may aid in assessing the hemodynamic significance of coronary artery disease.

  18. Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Mahmood, R.

    1985-01-01

    The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz 2 ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz 2 ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF 1 ) with a binding constant of 3.2 x 10 5 M -1 . Bz 2 ATP is also a substrate for the ATPase activity of SF 1 in the presence of different cations. The irradiation of SF 1 with [ 3 H]Bz 2 ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF 1 after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling [ 3 H]Bz 2 ATP is trapped on SF 1 by cross-linking the two reactive thiols, SH 1 and SH 2 , by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified [ 14 C]Bz 2 ATP-SF 1 , after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography

  19. A novel fluorescent biosensor for Adenosine Triphosphate detection based on the polydopamine nanospheres integrating with enzymatic recycling amplification.

    Science.gov (United States)

    Ji, Xiaoting; Yi, Bingqing; Xu, Yujuan; Zhao, Yanan; Zhong, Hua; Ding, Caifeng

    2017-07-01

    Based on the protective performance of polydopamine nanospheres (PDANSs) for DNA against nuclease digestion and the specific recognition characteristic of aptamer, we have developed an enzymatic recycling signal amplification method for highly sensitive and selective detection of adenosine triphosphate (ATP). Fluorescence measurements were carried out to verify the DNA polymerase and exonuclease III (Exo III) assisted target recycling process and fluorescence signal amplification. In the absence of the ATP, initially, the signal DNA-PDANSs complex was in the "off" state due to the efficient fluorescence quenching of 6-carboxyfluorescein (FAM) adjacent to the surface of PDANSs. Due to the binding of the aptamer by ATP, it trigger DNA polymerase and Exo III assisted target recycling process by the product of release, the complex would change into the "on" state as a result of the dissociation of the FAM from the surface of PDANSs, thus providing greatly enhanced fluorescence emission intensity. The method allows quantitative detection of ATP in the range of 20-600nM with a detection limit of 8.32nM. This biosensor requires no complex operations, and is a new high efficiency method for ATP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Spectroscopic study of the interaction between adenosine disodium triphosphate and gatifloxacin-Al3+ complex and its analytical application.

    Science.gov (United States)

    Kamruzzaman, Mohammad; Faruqui, A Nayeem; Hossain, Mohammed Ifteker; Lee, Sang Hak

    2015-11-01

    A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)-aluminium (III) (Al(3+) ) complex and ATP and studied using UV-visible and fluorescence spectroscopy. Weak luminescence spectra of Al(3+) were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al(3+) ion. It was observed that the FL emission spectrum of GFLX-Al(3+) was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10(-10) - 1.0 × 10(-8) mol L(-1) with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10(-11) mol L(-1) for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10(-8) mol L(-1) ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Heterogeneity of tumor chemosensitivity in ovarian epithelial cancer revealed using the adenosine triphosphate-tumor chemosensitivity assay.

    Science.gov (United States)

    Zhang, Jin; Li, Hongxia

    2015-05-01

    Ovarian cancer has a poor prognosis, primarily due to the heterogeneity in chemosensitivity among patients. In the present study, this heterogeneity was evaluated in ovarian epithelial cancer (OEC) using an in vitro adenosine triphosphate tumor chemosensitivity assay (ATP-TCA). Specimens were collected from 80 patients who underwent cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissues were tested for sensitivity to paclitaxel (PTX), carboplatin (CBP), topotecan (TPT), gemcitabine (GEM), docetaxel (TXT), etoposide, bleomycin and 4-hydroperoxycyclophosphamide using ATP-TCA. The sensitivity, specificity, positive predictive value and negative predictive value for the clinical chemotherapy sensitivity of OEC were 88.6, 77.8, 83 and 84.8%, respectively. PTX demonstrated the highest sensitivity of all agents tested (82.5% in all specimens, 85.7% in recurrent specimens), followed by CBP (58.8 and 60.7%, respectively). The sensitivities to PTX and docetaxel (PIII) or low-differentiated specimens, respectively. The present study indicated that ATP-TCA is an effective method for guiding the choice of chemotherapy drugs. Notable heterogeneity of chemosensitivity was observed in the OEC specimens.

  2. Adsorption characteristics of 14C-labeled alanine, aspartic acid and adenosine triphosphate by metal-chelating resins

    International Nuclear Information System (INIS)

    Ishiyama, Toshio; Matsunami, Tadao; Shibata, Setsuko; Honda, Yoshihide.

    1987-01-01

    (1) Adsorption properties of 14 C-alanine, 14 C-ATP (adenosine triphosphate) and 14 C-aspartic acid on the metal-chelating resins were determined and found that the Cu(II)-Chelex 100 and Fe(III)-Unicellex UR10, Fe(III)-Chelex 100 chelating resins were highly effective for the adsorption of 14 C-alanine and 14 C-ATP, respectively. (2) Desorption rate of 14 C-ATP from the Fe(III)-Unicellex UR10 and Fe(III)-Chelex 100 resins was somewhat higher than the case of 14 C-alanine, probably because the coordination bonds of Cu-alanine might be stronger than those of Fe-ATP. Thus, 14 C-labeled organic compounds such as 14 C-alanine and 14 C-ATP of a low activity concentration (3.7 mBq/ml) (1 x 10 -7 μCi/ml) in aqueous solution may be measured with liquid scintillation counter after pre-concentration by use of the Fe(III)- and Cu(II)-chelating resin columns. (author)

  3. Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

    Science.gov (United States)

    Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

    2014-07-15

    A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

  4. Kinetic, spectroscopic and chemical modification study of iron release from transferrin; iron(III) complexation to adenosine triphosphate

    International Nuclear Information System (INIS)

    Thompson, C.P.

    1985-01-01

    Amino acids other than those that serve as ligands have been found to influence the chemical properties of transferrin iron. The catalytic ability of pyrophosphate to mediate transferrin iron release to a terminal acceptor is largely quenched by modification non-liganded histine groups on the protein. The first order rate constants of iron release for several partially histidine modified protein samples were measured. A statistical method was employed to establish that one non-liganded histidine per metal binding domain was responsible for the reduction in rate constant. These results imply that the iron mediated chelator, pyrophosphate, binds directly to a histidine residue on the protein during the iron release process. EPR spectroscopic results are consistent with this interpretation. Kinetic and amino acid sequence studies of ovotransferrin and lactoferrin, in addition to human serum transferrin, have allowed the tentative assignment of His-207 in the N-terminal domain and His-535 in the C-terminal domain as the groups responsible for the reduction in rate of iron release. The above concepts have been extended to lysine modified transferrin. Complexation of iron(II) to adenosine triphosphate (ATP) was also studied to gain insight into the nature of iron-ATP species present at physiological pH. 31 P NMR spectra are observed when ATP is presented in large excess

  5. Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Shannon E.; Nguyen, Elaine; Ukachukwu, Chiamaka U.; Freeman, Dana M.; Quirk, Stephen; Lieberman, Raquel L.; Boggon, Titus J.

    2017-07-25

    Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

  6. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.

    Science.gov (United States)

    Ito, Mai; Arakawa, Toshiya; Okayama, Miki; Shitara, Akiko; Mizoguchi, Itaru; Takuma, Taishin

    2014-11-01

    The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells. Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation. Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells. Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement. © 2013 Wiley Publishing Asia Pty Ltd.

  7. Target-triggered transcription machinery for ultra-selective and sensitive fluorescence detection of nucleoside triphosphates in one minute.

    Science.gov (United States)

    Dong, Jiantong; Wu, Tongbo; Xiao, Yu; Chen, Lu; Xu, Lei; Li, Mengyuan; Zhao, Meiping

    2018-02-15

    Nucleoside triphosphates (NTPs) play important roles in living organisms. However, no fluorescent assays are currently available to simply and rapidly detect multiple NTPs with satisfactory selectivity, sensitivity and low cost. Here we demonstrate for the first time a target-triggered in-vitro transcription machinery for ultra-selective, sensitive and instant fluorescence detection of multiple NTPs. The machinery assembles RNA polymerase, DNA template and non-target NTPs to convert the target NTP into equivalent RNA signal sequences which are monitored by the fluorescence enhancement of molecular beacon. The machinery offers excellent selectivity for the target NTP against NDP, NMP and dNTP. Notably, to accelerate the kinetics of the machinery while maintain its high specificity, we investigated the sequence of DNA templates systematically and established a set of guidelines for the design of the optimum DNA templates, which allowed for instant detection of the target NTP at fmol level in less than 1min. Furthermore, the machinery could be transformed into logic gates to study the coeffects of two NTPs in biosynthesis and real-time monitoring systems to reflect the distribution of NTP in nucleotide pools. These results provide very useful and low-cost tools for both biochemical tests and point-of-care analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

    Science.gov (United States)

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr; Vunjak-Novakovic, Gordana

    2013-10-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

  9. Effects of oral adenosine-5'-triphosphate supplementation on athletic performance, skeletal muscle hypertrophy and recovery in resistance-trained men.

    Science.gov (United States)

    Wilson, Jacob M; Joy, Jordan M; Lowery, Ryan P; Roberts, Michael D; Lockwood, Christopher M; Manninen, Anssi H; Fuller, John C; De Souza, Eduardo O; Baier, Shawn M; Wilson, Stephanie Mc; Rathmacher, John A

    2013-09-22

    Currently, there is a lack of studies examining the effects of adenosine-5'-triphosphate (ATP) supplementation utilizing a long-term, periodized resistance-training program (RT) in resistance-trained populations. Therefore, we investigated the effects of 12 weeks of 400 mg per day of oral ATP on muscular adaptations in trained individuals. We also sought to determine the effects of ATP on muscle protein breakdown, cortisol, and performance during an overreaching cycle. The study was a 3-phase randomized, double-blind, and placebo- and diet-controlled intervention. Phase 1 was a periodized resistance-training program. Phase 2 consisted of a two week overreaching cycle in which volume and frequency were increased followed by a 2-week taper (Phase 3). Muscle mass, strength, and power were examined at weeks 0, 4, 8, and 12 to assess the chronic effects of ATP; assessment performance variables also occurred at the end of weeks 9 and 10, corresponding to the mid and endpoints of the overreaching cycle. There were time (psupplementation. During the overreaching cycle, there were group x time effects for strength and power, which decreased to a greater extent in the placebo group. Protein breakdown was also lower in the ATP group. Our results suggest oral ATP supplementation may enhance muscular adaptations following 12-weeks of resistance training, and prevent decrements in performance following overreaching. No statistically or clinically significant changes in blood chemistry or hematology were observed. ClinicalTrials.gov NCT01508338.

  10. Taste dysfunction in BTBR mice due to a mutation of Itpr3, the inositol triphosphate receptor 3 gene

    Science.gov (United States)

    Ellis, Hillary T.

    2013-01-01

    The BTBR T+ tf/J (BTBR) mouse strain is indifferent to exemplars of sweet, Polycose, umami, bitter, and calcium tastes, which share in common transduction by G protein-coupled receptors (GPCRs). To investigate the genetic basis for this taste dysfunction, we screened 610 BTBR × NZW/LacJ F2 hybrids, identified a potent QTL on chromosome 17, and isolated this in a congenic strain. Mice carrying the BTBR/BTBR haplotype in the 0.8-Mb (21-gene) congenic region were indifferent to sweet, Polycose, umami, bitter, and calcium tastes. To assess the contribution of a likely causative culprit, Itpr3, the inositol triphosphate receptor 3 gene, we produced and tested Itpr3 knockout mice. These were also indifferent to GPCR-mediated taste compounds. Sequencing the BTBR form of Itpr3 revealed a unique 12 bp deletion in Exon 23 (Chr 17: 27238069; Build 37). We conclude that a spontaneous mutation of Itpr3 in a progenitor of the BTBR strain produced a heretofore unrecognized dysfunction of GPCR-mediated taste transduction. PMID:23859941

  11. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  12. Heterogeneity of tumor chemosensitivity in ovarian epithelial cancer revealed using the adenosine triphosphate-tumor chemosensitivity assay

    Science.gov (United States)

    ZHANG, JIN; LI, HONGXIA

    2015-01-01

    Ovarian cancer has a poor prognosis, primarily due to the heterogeneity in chemosensitivity among patients. In the present study, this heterogeneity was evaluated in ovarian epithelial cancer (OEC) using an in vitro adenosine triphosphate tumor chemosensitivity assay (ATP-TCA). Specimens were collected from 80 patients who underwent cytoreductive surgery. Viable ovarian cancer cells obtained from malignant tissues were tested for sensitivity to paclitaxel (PTX), carboplatin (CBP), topotecan (TPT), gemcitabine (GEM), docetaxel (TXT), etoposide, bleomycin and 4-hydroperoxycyclophosphamide using ATP-TCA. The sensitivity, specificity, positive predictive value and negative predictive value for the clinical chemotherapy sensitivity of OEC were 88.6, 77.8, 83 and 84.8%, respectively. PTX demonstrated the highest sensitivity of all agents tested (82.5% in all specimens, 85.7% in recurrent specimens), followed by CBP (58.8 and 60.7%, respectively). The sensitivities to PTX and docetaxel (P<0.001) were correlated, in addition to those of CBP, TPT and GEM (P<0.001). Early-stage (I/II) and high- to mildly-differentiated OEC specimens revealed a lower chemosensitivity than advanced-stage (III) or low-differentiated specimens, respectively. The present study indicated that ATP-TCA is an effective method for guiding the choice of chemotherapy drugs. Notable heterogeneity of chemosensitivity was observed in the OEC specimens. PMID:26137074

  13. Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

    Science.gov (United States)

    Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

    2017-06-01

    In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

  14. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    Energy Technology Data Exchange (ETDEWEB)

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  15. Thrombin and PAF stimulate formation of inositol triphosphate from similar pools of phosphatidylinositol-4,5-bisphosphate in platelets

    International Nuclear Information System (INIS)

    Shukla, S.D.; Franklin, C.C.

    1986-01-01

    During platelet activation phospholipase C cleaves phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to generate diacylglycerol and inositol triphosphate (IP 3 ). Production of IP 3 was therefore used as a probe to differentiate between thrombin and platelet activating factor (PAF) sensitive pools of PIP 2 . Stimulation of [ 3 H]inositol labelled rabbit platelets (0.5 x 10 9 cells/ml containing 0.1 mM EGTA and 2 mM LiCl) with thrombin (1 U/ml) caused a maximum 3 to 10 fold increase in [ 3 H]IP 3 in 5 min. Treatment with PAF (1 x 10 -9 M) led to a maximum 3 to 5 fold increase in [ 3 H]IP 3 in 5 s followed by a decrease to basal level in 30 s. Addition of thrombin for 5 min followed by PAF for 5 s showed no further increase in [ 3 H]IP 3 over that obtained with thrombin alone. Various other sequential treatments with thrombin and PAF showed negligible cumulative effect. Simultaneous addition of thrombin and PAF showed a partial additive production of [ 3 H]IP 3 at short time periods but these levels never significantly exceeded those obtained by thrombin at 5 min or PAF at 5 s. It is concluded that in platelets thrombin and PAF (a) differentially activate PIP 2 -phosphodiesterase and (b) stimulate production of IP 3 from similar pools of PIP 2

  16. Potentiation of adenosine triphosphate-induced contractile responses of the guinea-pig isolated vas deferens by adenosine monophosphate and adenosine 5'-monophosphorothioate.

    OpenAIRE

    Fedan, J. S.

    1987-01-01

    The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (3...

  17. Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

    Science.gov (United States)

    Hocek, Michal

    2014-11-07

    The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

  18. Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction.

    Science.gov (United States)

    Silva, Isabel; Ferreirinha, Fátima; Magalhães-Cardoso, Maria Teresa; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

    2015-10-01

    Deregulation of purinergic bladder signaling may contribute to persistent detrusor overactivity in patients with bladder outlet obstruction. Activation of uridine diphosphate sensitive P2Y6 receptors increases voiding frequency in rats indirectly by releasing adenosine triphosphate from the urothelium. To our knowledge this mechanism has never been tested in the human bladder. We examined the role of the uridine diphosphate sensitive P2Y6 receptor on tetrodotoxin insensitive nonneuronal adenosine triphosphate and [(3)H]acetylcholine release from the human urothelium with the lamina propria of control organ donors and patients with benign prostatic hyperplasia. The adenosine triphosphate-to-[(3)H]acetylcholine ratio was fivefold higher in mucosal urothelium/lamina propria strips from benign prostatic hyperplasia patients than control men. The selective P2Y6 receptor agonist PSB0474 (100 nM) augmented by a similar amount adenosine triphosphate and [(3)H]acetylcholine release from mucosal urothelium/lamina propria strips from both groups of individuals. The facilitatory effect of PSB0474 was prevented by MRS2578 (50 nM) and by carbenoxolone (10 μM), which block P2Y6 receptor and pannexin-1 hemichannels, respectively. Blockade of P2X3 (and/or P2X2/3) receptors with A317491 (100 nM) also attenuated release facilitation by PSB0474 in control men but not in patients with benign prostatic hyperplasia. Immunolocalization studies showed that P2Y6, P2X2 and P2X3 receptors were present in choline acetyltransferase positive urothelial cells. In contrast to P2Y6 staining, choline acetyltransferase, P2X2 and P2X3 immunoreactivity decreased in the urothelium of benign prostatic hyperplasia patients. Activation of P2Y6 receptor amplifies mucosal adenosine triphosphate release underlying bladder overactivity in patients with benign prostatic hyperplasia. Therefore, we propose selective P2Y6 receptor blockade as a novel therapeutic strategy to control persistent storage symptoms in

  19. Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

    Science.gov (United States)

    Lin, Jia-Hui; Yang, Ya-Chun; Shih, Ya-Chen; Hung, Szu-Ying; Lu, Chi-Yu; Tseng, Wei-Lung

    2016-03-15

    Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

    Directory of Open Access Journals (Sweden)

    Jerome Deval

    2015-06-01

    Full Text Available Respiratory syncytial virus (RSV causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV, whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

  1. Depletion of cellular adenosine triphosphate and hepatocellular damage in rat after subchronic exposure to leachate from anthropogenic recycling site.

    Science.gov (United States)

    Akintunde, J K; Oboh, G

    2015-11-01

    One of the major hazards arising from recycling sites is the generation of leachate containing mixed metal. This study evaluated the toxic effects of leachate obtained from Elewi Odo municipal auto-battery recycling site (EOMABRSL) on male liver functions using hepatic indices and biomarker of cellular adenosine triphosphate (ATP) in rat via the oral route. Concentrations of heavy metals analysis showed that lead, cadmium, nickel, chromium, manganese, and iron were 1.5-, 2-, 2.5-, 1.36-, 19.61-, and 8.89-folds, respectively, higher than acceptable limits set by regulatory authority World Health Organization. Copper, zinc, and cobalt were 5.9-, 300-, and 1.02-folds, respectively, lower than permissible limits. The EOMABRSL was administered at 20, 40, 60, 80, and 100% concentrations to adult male rats for 60 days. Following exposure, plasma and livers were collected for several biochemistry assays. Exposure of animals to EOMABRSL resulted in 27.51, 28.14, 63.93, 28.42, and 40.16% increase in aspartate aminotransferase activity, whereas it elevated alanine aminotransferase activity by 5.35, 22.33, 88.68, 183.02, and 193.08%, respectively, when compared with the control. Similarly, γ-glutamyl transferase activity increased by 111.22, 114.19, 122.96, 573.14, and 437.02%, respectively, when compared with the control. EOMABRSL administration significantly decreased catalase activity and reduced glutathione level and superoxide dismutase with concomitant increase in malondialdehyde and hydrogen peroxide levels. Also, significant (p health hazards in subjects with occupational or environmental exposure. © The Author(s) 2015.

  2. Comparison of myocardial blood flow induced by adenosine triphosphate and dipyridamole in patients with coronary artery disease

    International Nuclear Information System (INIS)

    Mamede, M.; Tadamura, Eiji; Hosokawa, Ryohei

    2005-01-01

    Myocardial perfusion imaging with adenosine triphosphate (ATP) has been used increasingly to diagnose coronary artery disease (CAD) and assess risk for this disease. This study compared absolute myocardial blood flow (MBF) and myocardial flow reserve index (MFR) with ATP and dipyridamole (DIP) in patients with CAD. MBF was quantified by 15 O-H 2 O PET in 21 patients with CAD (17 male, 4 female), aged 55 to 81 years. MBF was measured at rest, during intravenous injection of ATP (0.16 mg/kg/min), and again after DIP infusion (0.56 mg/kg). Regions of interest were drawn in nonischemic and ischemic segments based on findings from thallium-201 ( 201 Tl) scintigraphy and coronary angiography (CAG). Absolute MBF values and indexes of MFR were calculated in nonischemic and ischemic segments. Intravenous injection of ATP and DIP significantly increased MBF in nonischemic (2.4±0.9 and 2.1±0.8 ml/g/min, respectively; p<0.01, for both) and in ischemic segments (1.3±0.4 and 1.5±0.4 ml/g/min, respectively; p<0.01, for both). There was a significant difference in MBF values between ATP and DIP in nonischemic segments (p<0.05), which was not observed in ischemic segments. In nonischemic segments, ATP produced higher MFR than DIP (2.1±0.8 and 1.8±0.7, respectively; p<0.05), while no significant difference was observed in ischemic segments (1.5±0.6 and 1.7±0.3, respectively). ATP produced a greater hyperemia than DIP between the ischemic and nonischemic myocardium in patients with CAD. ATP is as effective as DIP for the diagnosis of CAD. (author)

  3. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases. cap alpha. and delta by butylphenyl deoxyguanosine triphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Dreslor, S.L.; Frattini, M.G.

    1987-05-01

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, ..cap alpha.. and/or delta. They have studied the inhibition of replication and repair synthesis in permeable human cells by N/sup 2/ (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase ..cap alpha.. strongly and polymerase delta weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 ..mu..M and, for repair synthesis, 3-4 ..mu..M, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase ..cap alpha.. by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase delta is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase delta were hampered by the finding that the dependence of delta activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase ..cap alpha.. and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase delta, but some other characteristics of the cellular processes are more similar to those of polymerase ..cap alpha...

  4. Adenosine Triphosphate (ATP Is a Candidate Signaling Molecule in the Mitochondria-to-Nucleus Retrograde Response Pathway

    Directory of Open Access Journals (Sweden)

    Zhengchang Liu

    2013-03-01

    Full Text Available Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to and inactivates Mks1, allowing for activation of Rtg1/3 and the RTG pathway. When the pathway is inactive, Mks1 has dissociated from Rtg2 and bound to Bmh1/2, preventing activation of Rtg1/3. What signals association or disassociation of Mks1 and Rtg2 is unknown. Here, we show that ATP at physiological concentrations dissociates Mks1 from Rtg2 in a highly cooperative fashion. We report that ATP-mediated dissociation of Mks1 from Rtg2 is conserved in two other fungal species, K. lactis and K. waltii. Activation of Rtg1/3 upregulates expression of genes encoding enzymes catalyzing the first three reactions of the Krebs cycle, which is coupled to ATP synthesis through oxidative phosphorylation. Therefore, we propose that the retrograde response is an ATP homeostasis pathway coupling ATP production with ATP-mediated repression of the retrograde response by releasing Mks1 from Rtg2.

  5. Effects of chlorogenic acid on adenine nucleotides hydrolyzing enzyme activities and expression in platelets of rats experimentally demyelinated with ethidium bromide.

    Science.gov (United States)

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; Beckmann, Diego V; Castilhos, Lívia G; Thorstenberg, Maria L P; Jaques, Jeandre A Dos S; Souza, Viviane do C G; Farias, Júlia G; Martins, Caroline C; Schetinger, Maria R C

    2016-07-01

    The effects of chlorogenic acid (one of the major phenolic acid found in human diets) were investigated on the adenine nucleotides hydrolyzing enzymes; ecto-nucleotide pyrophosphatase/phophodiesterase (E-NPP), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'- nucleotidase and ecto-adenosine deaminase (E-ADA) activities and expression in platelets of rats experimentally demyelinated with ethidium bromide. Rats were divided into four groups of eight animals each. Group I rats were control rats; injected with saline (CT), group II rats were injected with saline and treated with chlorogenic acid (AC), group III rats were injected with 0.1% ethidium bromide (EB) and group IV rats were injected with 0.1% EB and treated with chlorogenic acid (EB+AC). The activities of the enzymes were analyzed using colorimetric methods, and the gene expression of NTPDase 1, 2 and 3 were analyzed using the polymerase chain reaction (PCR). The results revealed that there was a significant (Pchlorogenic acid significantly (P0.05) change was observed in the E-NPP activity of EB+AC group (2.19±0.08nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33±0.14nmol p-nitrophenol released/min/mg protein). In addition, there was a significant (P0.05) difference was observed in AMP hydrolysis between AC, AC+EB and CT groups. Conversely, there were no significant (P>0.05) differences in ATP and ADP hydrolyses between all the groups (AC, EB, AC+EB and CT groups). Likewise, there were no significant (P>0.05) changes in E-ADA activity and percentage platelet aggregation among all groups studied. Similarly, no significant (P>0.05) change was observed in the expression of E-NTPDase 1, 2 and 3 in all the groups tested. Our study revealed that chlorogenic acid may modulate the hydrolysis of adenine nucleotides in platelets of rats demyelinated and treated with chlorogenic acid via alteration of E-NPP and ecto-5'-nucleotidase activities. Copyright © 2016 Elsevier

  6. Alkaloid extracts from Jimson weed (Datura stramonium L.) modulate purinergic enzymes in rat brain.

    Science.gov (United States)

    Ademiluyi, Adedayo O; Ogunsuyi, Opeyemi B; Oboh, Ganiyu

    2016-09-01

    Although some findings have reported the medicinal properties of Jimson weed (Datura stramonium L.), there exist some serious neurological effects such as hallucination, loss of memory and anxiety, which has been reported in folklore. Consequently, the modulatory effect of alkaloid extracts from leaf and fruit of Jimson weed on critical enzymes of the purinergic [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-NTDase), alkaline phosphatase (ALP) and Na + /K + ATPase] system of neurotransmission was the focus of this study. Alkaloid extracts were prepared by solvent extraction method and their interaction with the activities of these enzymes were assessed (in vitro) in rat brain tissue homogenate and in vivo in rats administered 100 and 200mg/kg body weight (p.o) of the extracts for thirty days, while administration of single dose (1mg/kg body weight; i.p.) of scopolamine served as the positive control. The extracts were also investigated for their Fe 2+ and Cu 2+ chelating abilities and GC-MS characterization of the extracts was also carried out. The results revealed that the extracts inhibited activates of E-NTPDase, E-NTDase and ALP in a concentration dependent manner, while stimulating the activity of Na + /K + ATPase (in vitro). Both extracts also exhibited Fe 2+ and Cu 2+ chelating abilities. Considering the EC 50 values, the fruit extract had significantly higher (P0.05) in both extracts' inhibitory effects on E-NTDase. The in vivo study revealed reduction in the activities of ENTPDase, E-NTDase, and Na + /K + ATPase in the extract-administered rat groups compared to the control group, while an elevation in ALP activity was observed in the extract-administered rat groups compared to the control group. GC-MS characterization revealed the presence of atropine, scopolamine, amphetamine, 3-methyoxyamphetamine, 3-ethoxyamhetamine cathine, spermine, phenlyephirine and 3-piperidinemethanol, among others in the extracts. Hence

  7. Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

    International Nuclear Information System (INIS)

    Ide, H.; Melamede, R.J.; Wallace, S.S.

    1987-01-01

    5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag 2 O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [ 3 H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The result suggests that Escherichia coli DNA polymerase I uses both isomers of DHdTTP as substrates and that the overall efficiency of incorporation is primarily determined by the concentration of the isomers in the nucleotide pool

  8. On the use of X-ray absorption spectroscopy to elucidate the structure of lutetium adenosine mono- and triphosphate complexes.

    Science.gov (United States)

    Mostapha, S; Berthon, C; Fontaine-Vive, F; Gaysinski, M; Guérin, L; Guillaumont, D; Massi, L; Monfardini, I; Solari, P L; Thomas, O P; Charbonnel, M C; Den Auwer, C

    2014-02-01

    Although the physiological impact of the actinide elements as nuclear toxicants has been widely investigated for half a century, a description of their interactions with biological molecules remains limited. It is however of primary importance to better assess the determinants of actinide speciation in cells and more generally in living organisms to unravel the molecular processes underlying actinide transport and deposition in tissues. The biological pathways of this family of elements in case of accidental contamination or chronic natural exposure (in the case of uranium rich soils for instance) are therefore a crucial issue of public health and of societal impact. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, phosphate derivatives are considered as probable targets of these cations. Among them, nucleotides and in particular adenosine mono- (AMP) and triphosphate (ATP) nucleotides occur in more chemical reactions than any other compounds on the earth's surface, except water, and are therefore critical target molecules. In the present study, we are interested in trans-plutonium actinide elements, in particular americium and curium that are more rarely considered in environmental and bioaccumulation studies than early actinides like uranium, neptunium and plutonium. A first step in this strategy is to work with chemical analogues like lanthanides that are not radioactive and therefore allow extended physical chemical characterization to be conducted that are difficult to perform with radioactive materials. We describe herein the interaction of lutetium(III) with adenosine AMP and ATP. With AMP and ATP, insoluble amorphous compounds have been obtained with molar ratios of 1:2 and 1:1, respectively. With an excess of ATP, with 1:2 molar ratio, a soluble complex has been obtained. A combination of spectroscopic techniques (IR, NMR, ESI-MS, EXAFS) together with quantum

  9. Extracellular adenosine 5'-triphosphate concentrations changes in rat spinal cord associated with the activation of urinary bladder afferents. A microdialysis study.

    Science.gov (United States)

    Rocha, Jeová Nina

    2016-01-01

    To determine adenosine 5'-triphosphate levels in the interstice of spinal cord L6-S1 segment, under basal conditions or during mechanical and chemical activation of urinary bladder afferents. A microdialysis probe was transversally implanted in the dorsal half of spinal cord L6-S1 segment in female rats. Microdialysate was collected at 15 minutes intervals during 135 minutes, in anesthetized animals. Adenosine 5'-triphosphate concentrations were determined with a bioluminescent assay. In one group of animals (n=7) microdialysate samples were obtained with an empty bladder during a 10-minutes bladder distension to 20 or 40cmH2O with either saline, saline with acetic acid or saline with capsaicin. In another group of animals (n=6) bladder distention was performed and the microdialysis solution contained the ectonucleotidase inhibitor ARL 67156. Basal extracellular adenosine triphosphate levels were 110.9±35.34fmol/15 minutes, (mean±SEM, n=13), and bladder distention was associated with a significant increase in adenosine 5'-triphosphate levels which was not observed after bladder distention with saline solution containing capsaicin (10µM). Microdialysis with solution containing ARL 67156 (1mM) was associated with significantly higher extracellular adenosine 5'-triphosphate levels and no further increase in adenosine 5'-triphosphate was observed during bladder distension. Adenosine 5'-triphosphate was present in the interstice of L6-S1 spinal cord segments, was degraded by ectonucleotidase, and its concentration increased following the activation of bladder mechanosensitive but not of the chemosensitive afferents fibers. Adenosine 5'-triphosphate may originate either from the central endings of bladder mechanosensitive primary afferent neurons, or most likely from intrinsic spinal neurons, or glial cells and its release appears to be modulated by capsaicin activated bladder primary afferent or by adenosine 5'-triphosphate itself. Determinar as concentra

  10. Increased Na+/K(+)-pump activity and adenosine triphosphate utilization after compound 48/80-induced histamine secretion from rat mast cells

    DEFF Research Database (Denmark)

    Johansen, Torben; Praetorius, Birger Hans

    1994-01-01

    The Na+/K(+)-pump activity and the utilization of adenosine triphosphate (ATP) were studied in rat peritoneal mast cells after histamine secretion induced by compound 48/80. We measured the ouabain-sensitive K(+)-uptake by a radioactive technique (86Rb+). The ATP content and the glycolytic ATP...... was the limiting factor for the activity of the Na+/K(+)-pump following histamine secretion under these conditions. It is concluded that the large increase in Na+/K(+)-pump activity after a secretory response is a likely explanation for the long lasting ATP-decrease in mast cells that follows histamine secretion....

  11. [Adenosine triphosphate stress 99mTc-MIBI single-photon emission computed tomography in the diagnosis of ischemic heart disease].

    Science.gov (United States)

    Karpova, I E; Samoĭlenko, L E; Soboleva, G N; Sergienko, V B; Karpov, Iu A

    2013-01-01

    This review is devoted to possibilities of single-photon emission computed tomography (SPECT) combined with pharmacological test with adenosine triphosphate (ATP) to detect myocardial ischemia in patients with ischemic heart disease (IHD). It contains consideration of contemporary problems and limitations inherent in use of pharmacological stress tests in radionuclide diagnostics; discussion of mechanisms of vasodilating effects of ATP in the context of modern concepts of purine receptors; detailed description of technique of pharmacological testing with ATP, as well as contraindications and possible side effects. Experience of foreign studies with the use of ATP stress testing for verification of presence of ischemia in patients with IHD is also presented.

  12. Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios

    Directory of Open Access Journals (Sweden)

    Dorris David

    2002-07-01

    Full Text Available Abstract Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. Conclusions Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.

  13. Capillary electrophoresis coupled to mass spectrometry employing hexafluoro-2-propanol for the determination of nucleosides and nucleotide mono-, di- and tri-phosphates in baby foods.

    Science.gov (United States)

    Mateos-Vivas, María; Domínguez-Álvarez, Javier; Rodríguez-Gonzalo, Encarnación; Carabias-Martínez, Rita

    2017-10-15

    The present work describes a method for the simultaneous determination of unmodified nucleosides and nucleotide mono-, di- and tri-phosphates by capillary electrophoresis coupled to mass spectrometry (CE-MS). The use of hexafluoro-2-propanol (HFIP) in the separation medium, and as an additive to the sheath liquid of the electrospray interface (ESI), generated a highly efficient and sensitive method. Instrumental limits of detection in the range of 14-53ngmL -1 for nucleosides and 7-23, 20-49 and 64-124ngmL -1 for nucleotide mono-, di-, and tri-phosphates, respectively, were found. Sample treatment involved diluting an aliquot of baby food with ultra-high quality water and applying centrifugation-assisted ultrafiltration (CUF). The proposed method was validated and used to analyse a variety of baby food samples (16 in total) such as fish, meat, fruits, and baby dairy desserts that may endogenously contain these analytes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Simple, Fast and Selective Detection of Adenosine Triphosphate at Physiological pH Using Unmodified Gold Nanoparticles as Colorimetric Probes and Metal Ions as Cross-Linkers

    Directory of Open Access Journals (Sweden)

    Huan Pang

    2012-11-01

    Full Text Available We report a simple, fast and selective colorimetric assay of adenosine triphosphate (ATP using unmodified gold nanoparticles (AuNPs as probes and metal ions as cross-linkers. ATP can be assembled onto the surface of AuNPs through interaction between the electron-rich nitrogen atoms and the electron-deficient surface of AuNPs. Accordingly, Cu2+ ions induce a change in the color and UV/Vis absorbance of AuNPs by coordinating to the triphosphate groups and a ring nitrogen of ATP. A detection limit of 50 nM was achieved, which is comparable to or lower than that achievable by the currently used electrochemical, spectroscopic or chromatographic methods. The theoretical simplicity and high selectivity reported herein demonstrated that AuNPs-based colorimetric assay could be applied in a wide variety of fields by rationally designing the surface chemistry of AuNPs. In addition, our results indicate that ATP-modified AuNPs are less stable in Cu2+, Cd2+ or Zn2+-containing solutions due to the formation of the corresponding dimeric metal-ATP complexes.

  15. Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts

    International Nuclear Information System (INIS)

    Kaplan, J.H.; Forbush, B. III; Hoffman, J.F.

    1978-01-01

    2-Nitrobenzyl phosphate and 1-(2-nitro)phenylethyl phosphate have been synthesized and demonstrated to be suitable as photolabile sources of inorganic phosphate. The same protecting groups were attached to the terminal phosphate of adenosine 5'-triphosphate. These caged ATP compounds released adenosine 5'-triphosphate on illumination at 340 nm in aqueous solution and P 3 -1-(2-nitro)phenylethyl-ATP gave about a 70 percent yield in under 30 s. The unphotolyzed caged ATP was neither a substrate nor inhibitor of purified renal Na,K-ATPase (EC 3.61.3). Following photolysis in the presence of the enzyme, the liberated ATP was hydrolyzed but at an inhibited rate. The photo-dependent inhibition could be eliminated by prior addition of glutathione or bisulfite to the irradiated solution. Caged ATP was incorporated into resealed human erythrocyte ghosts prepared from red blood cells depleted of internal energy stores. While the NA : K pump was unable to use incorporated caged ATP as a substrate, the ATP liberated by photolysis activated the pump as evidenced by measurements of K-dependent, ouabain-sensitive Na efflux. Thus the caged ATP can be used as a stable source of ATP unmetabolizable by intracellular ATPases until the ATP is released following photolytic irradiation

  16. Prolonged Atrioventricular Block and Ventricular Standstill Following Adenosine Triphosphate Injection in a Patient Taking Dipyridamole and Antiarrhythmic Agents: A Case Report

    Directory of Open Access Journals (Sweden)

    Kotaro Oe, MD

    2009-01-01

    Full Text Available An 83-year-old woman was admitted to our hospital because of palpitation. She had hypertension and paroxysmal atrial fibrillation, treated with digoxin and cibenzoline, and took dipyridamole for microalbuminuria. Before admission, she had taken pilsicainide pills in addition. On admission, electrocardiogram showed regular tachycardia with mildly prolonged QRS width. For the purpose of terminating tachycardia, 10 mg of adenosine triphosphate (ATP was rapidly injected. About 20 sec later, atrioventricular block and ventricular standstill occurred. She presented loss of consciousness and convulsion, and chest compression was performed. About 30 sec later, the QRS complex reappeared, and she became alert. Serum concentration of digoxin, cibenzoline and pilsicainide was within therapeutic level, respectively. We should be cautious in using ATP for a patient taking dipyridamole and antiarrhythmic agents.

  17. The effect of growth phase and medium on the use of the firefly adenosine triphosphate (ATP) assay for the quantitation of bacteria

    Science.gov (United States)

    Bush, V. N.; Picciolo, G. L.; Chappelle, E. W.

    1975-01-01

    Luciferase assay for adenosine triphosphate (ATP) was used as a rapid method to determine the number of bacteria in a urine sample after nonbacterial components were removed. Accurate cellular ATP values, determined when bacteria were grown in an environment similar to that in which they were found, were necessary for the calculation of bacterial titer in urine. Cellular ATP values vary depending on the extraction method, the cell growth phase, and cell growth conditions. ATP per cell values of stationary E. coli grown in urine were two times greater than ATP per cell values of cells grown in trypticase soy broth. Glucose and urea were examined as possible components responsible for the cellular ATP variation.

  18. Effects of 5 Thio-D-Glucose on cellular adenosine triphosphate levels and deoxyribonucleic acid rejoining in hypoxic and aerobic Chinese hamster cells

    International Nuclear Information System (INIS)

    Nagle, W.A.; Moss, A.J. Jr.; Roberts, H.G. Jr.; Baker, M.L.

    1980-01-01

    Intracellular adenosine triphosphate (ATP) levels were measured in both hypoxic and aerobic cultures of V79 Chinese hamster cells treated with 5-thio-D-glucose (5-SH-D-Glc). This glucose analog, a known inhibitor of D-glucose transport and metabolism, reduced ATP in cell cultures allowed to become hypoxic by cell metabolism, but not in aerobic cultures treated similarly. Cells depleted of ATP were unable to rejoin x-ray induced deoxyribonucleic acid (DNA) strand breaks as measured by the alkaline sucrose gradient sedimentation technique. The inference for radiation therapy is that inhibition of glucose metabolism selectively depletes energy reserves in hypoxic cells, rendering these cells more radiosensitive and leading to a more effective tumor treatment

  19. Increased Na+/K(+)-pump activity and adenosine triphosphate utilization after compound 48/80-induced histamine secretion from rat mast cells

    DEFF Research Database (Denmark)

    Johansen, Torben; Praetorius, Birger Hans

    1994-01-01

    The Na+/K(+)-pump activity and the utilization of adenosine triphosphate (ATP) were studied in rat peritoneal mast cells after histamine secretion induced by compound 48/80. We measured the ouabain-sensitive K(+)-uptake by a radioactive technique (86Rb+). The ATP content and the glycolytic ATP......-production were measured by the bioluminescence technique (firefly lantern) and by measurement of the lactate production under anaerobic conditions (antimycin A, oligomycin), respectively. There was an increased requirement for ATP after the secretory response associated with an increased activity of the Na+/K(+)-pump....... The anaerobic, but not the aerobic, pathway for ATP-synthesis was able to respond to the increased ATP-requirement. The ATP-requirement of the Na+/K(+)-pump was only partly satisfied when ATP was supplied from either the glycolytic or the oxidative pathway. This may indicate that the availability of ATP...

  20. Molecular identification of Pomacea canaliculata and P. insularum from rice paddy in different origins in China using mitochondrial adenosine triphosphate subunit 6 gene.

    Science.gov (United States)

    Bian, Qing-Qing; Li, Xiao-Yan; Fang, Yan-Qin; Jia, Yan-Qing; Mu, Xi-Dong

    2015-02-01

    To identify Pomacea canaliculata and P. insularum using a molecular approach, the partial sequences of mitochondrial (mt) adenosine triphosphate subunit 6 (patp6) genes of two apple snails species from eight provinces of China were obtained. The intra-specific variations in mt patp6 within P. canaliculata were 0-4.5%, and no sequence difference in this region was observed within P. insularum. However, high inter-specific variations between P. canaliculata and P. insularum were detected, with sequence differences of 8.9-10.1%. Phylogenetic analyses based on sequences of mt patp6 revealed that P. canaliculata and P. insularum were grouped in different clades, but the genetic trees could not reveal geographically genetic relationships of P. canaliculata isolates from different origins. These findings will provide basic information for further study of molecular epidemiology and control of Pomacea invasion in China as well as in the world.

  1. Synthesis of 3'-azido and 3'-aminoarabinonucleoside 5'-triphosphates and study of their substrate properties in systems with polynucleotide-synthesizing enzymes

    International Nuclear Information System (INIS)

    Papchikhin, A.V.; Purygin, P.P.; Azhaev, A.V.; Kraevskii, A.A.; Kutateladze, T.V.; Chidzhavadze, Z.G.; Bibilashvili, R.Sh.

    1986-01-01

    5'-Triphosphates of 3'-azido- and 3'-amino-3'-deoxyarabinonucleosides containing adenine, guanine, cytosine, and thymine residues have been synthesized. Their substrate properties in cell-free systems with E. coli DNA polymerase I, calf thymus DNA polymerase α, phage T4 DNA polymerase, reverse transcriptase coded by the avian myeloblastosis virus, and also calf thymus terminal deoxynucleotidyl transferase have been studied. It has been shown that aNTP(3'NH 2 ) and aNTP(3'-N 3 ), act as DNA-chain-terminating substrates for DNA polymerase α, phage T4 DNA polymerase, reverse transcriptase, and terminal transferase. On the other hand, DNA polymerase I makes practically no use of these compounds as substrates

  2. SIMULTANEOUS ANALYSIS OF AZIDOTHYMIDINE AND ITS MONOPHOSPHATE, DIPHOSPHATE AND TRIPHOSPHATE DERIVATIVES IN BIOLOGICAL-FLUIDS, TISSUE AND CULTURED-CELLS BY A RAPID HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD

    NARCIS (Netherlands)

    MOLEMA, G; JANSEN, RW; Visser, Jan; MEIJER, DKF

    1992-01-01

    A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro

  3. Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template.

    Science.gov (United States)

    Nguyen, Laura A; Domaoal, Robert A; Kennedy, Edward M; Kim, Dong-Hyun; Schinazi, Raymond F; Kim, Baek

    2015-03-01

    Non-dividing macrophages maintain extremely low cellular deoxyribonucleotide triphosphate (dNTP) levels, but high ribonucleotide triphosphate (rNTP) concentrations. The disparate nucleotide pools kinetically forces Human Immunodeficiency Virus 1 (HIV-1) reverse transcriptase (RT) to incorporate non-canonical rNTPs during reverse transcription. HIV-1 RT pauses near ribonucleoside monophosphates (rNMPs) embedded in the template DNA, which has previously been shown to enhance mismatch extension. Here, pre-steady state kinetic analysis shows rNTP binding affinity (Kd) of HIV-1 RT for non-canonical rNTPs was 1.4- to 43-fold lower, and the rNTP rate of incorporation (kpol) was 15- to 1551-fold slower than for dNTPs. This suggests that RT is more selective for incorporation of dNTPs rather than rNTPs. HIV-1 RT selectivity for dNTP versus rNTP is the lowest for ATP, implying that HIV-1 RT preferentially incorporates ATP when dATP concentration is limited. We observed that incorporation of a dNTP occurring one nucleotide before an embedded rNMP in the template had a 29-fold greater Kd and a 20-fold slower kpol as compared to the same template containing dNMP. This reduced the overall dNTP incorporation efficiency of HIV-1 RT by 581-fold. Finally, the RT mutant Y115F displayed lower discrimination against rNTPs due to its increase in binding affinity for non-canonical rNTPs. Overall, these kinetic results demonstrate that HIV-1 RT utilizes both substrate binding and a conformational change during: (1) enzymatic discrimination of non-canonical rNTPs from dNTPs and (2) during dNTP primer extension with DNA templates containing embedded rNMP. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Tween 20-stabilized gold nanoparticles combined with adenosine triphosphate-BODIPY conjugates for the fluorescence detection of adenosine with more than 1000-fold selectivity

    International Nuclear Information System (INIS)

    Hung, Szu-Ying; Shih, Ya-Chen; Tseng, Wei-Lung

    2015-01-01

    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

  5. Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA

    International Nuclear Information System (INIS)

    Evans, R.K.; Haley, B.E.

    1987-01-01

    A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N 3 dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N 3 dUTP is the nitration of dUMP in 98% yield in 5 min at 25 0 C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH 2 dUMP). Diazotization of 5-NH 2 dUMP with HNO 2 followed by the addition NaN 3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N 3 dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N 3 dUTP through a chemical coupling of pyrophosphate to 5-N 3 dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N 3 UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [γ- 32 P]-5-N 3 dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing γ- 32 P-labeled pyrimidine nucleotides was developed. [γ- 32 P]-5-N 3 dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP. The photoactivity of 5-N 3 dUMP is stable to extremes of pH, and [γ- 32 P]-5-N 3 dUTP was an effective photolabeling reagent even in the presence of 10 mM dithiothreitol

  6. Effect of protonation on the mechanism of phosphate monoester hydrolysis and comparison with the hydrolysis of nucleoside triphosphate in biomolecular motors.

    Science.gov (United States)

    Hassan, Hammad Ali; Rani, Sadaf; Fatima, Tabeer; Kiani, Farooq Ahmad; Fischer, Stefan

    2017-11-01

    Hydrolysis of phosphate groups is a crucial reaction in living cells. It involves the breaking of two strong bonds, i.e. the O a H bond of the attacking water molecule, and the PO l bond of the substrate (O a and O l stand for attacking and leaving oxygen atoms). Mechanism of the hydrolysis reaction can proceed either by a concurrent or a sequential mechanism. In the concurrent mechanism, the breaking of O a H and PO l bonds occurs simultaneously, whereas in the sequential mechanism, the O a H and PO l bonds break at different stages of the reaction. To understand how protonation affects the mechanism of hydrolysis of phosphate monoester, we have studied the mechanism of hydrolysis of protonated and deprotonated phosphate monoester at M06-2X/6-311+G**//M06-2X/6-31+G*+ZPE level of theory (where ZPE stands for zero point energy). Our calculations show that in both protonated and deprotonated cases, the breaking of the water O a H bond occurs before the breaking of the PO l bond. Because the two events are not separated by a stable intermediate, the mechanism can be categorized as semi-concurrent. The overall energy barrier is 41kcalmol -1 in the unprotonated case. Most (5/6th) of this is due to the initial breaking of the water O a H bond. This component is lowered from 34 to 25kcalmol -1 by adding one proton to the phosphate. The rest of the overall energy barrier comes from the subsequent breaking of the PO l bond and is not sensitive to protonation. This is consistent with previous findings about the effect of triphosphate protonation on the hydrolysis, where the equivalent protonation (on the γ-phosphate) was seen to lower the barrier of breaking the water O a H bond and to have little effect on the PO l bond breaking. Hydrolysis pathways of phosphate monoester with initial breaking of the PO l bond could not be found here. This is because the leaving group in phosphate monoester cannot be protonated, unlike in triphosphate hydrolysis, where protonation of the

  7. Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

    Directory of Open Access Journals (Sweden)

    Satoshi Obika

    2010-11-01

    Full Text Available Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

  8. Activation of guanine-{beta}-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

    Energy Technology Data Exchange (ETDEWEB)

    Leanza, Luigi; Miazzi, Cristina; Ferraro, Paola; Reichard, Peter; Bianchi, Vera, E-mail: vbianchi@bio.unipd.it

    2010-12-10

    The deoxyguanosine (GdR) analog guanine-ss-D-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

  9. Identification of separate receptors for adenosine and adenosine 5'-triphosphate in causing relaxations of the isolated taenia of the guinea-pig caecum.

    Science.gov (United States)

    Spedding, M; Weetman, D F

    1976-01-01

    1 The mechanisms by which adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and adenosine relax the taenia caecum preparation of the guineapig have been studied. ATP and ADP produced similar effects which were qualitatively different from those of AMP and adenosine. 2 2-2'Pyridylisatogen tosylate (PIT: 50 muM for 30 min) blocked the effects of ATP and ADP, but exhibited weak activity against AMP and failed to antagonize the effects of adenosine. The action of PIT was unaffected by the inclusion of dipyridamole (2muM) in the bathing fluid. 3 There was a significant correlation between the sensitivity of individual preparations to ATP or ADP and the blocking potency of PIT. 4 The presence of adenosine in the bathing fluid (2 mM for greater than 30 min) desensitized the taenia to subsequent applications of adenosine. The effects of ATP were increased by this procedure. 5 The results indicate that ATP and adenosine relax the taenia by different mechanisms. PMID:938799

  10. Interaction centres of purine nucleotides: adenosine-5'-diphosphate and adenosine-5'-triphosphate in their reactions with tetramines and Cu(II) ions.

    Science.gov (United States)

    Gasowska, A

    2003-08-01

    The interactions between the nucleotides: adenosine-5'-diphosphate (ADP) and adenosine-5'-triphosphate (ATP) with spermine (Spm) and 1,11-diamine-4,8-diazaundecane (3,3,3-tet), as well as Cu(II) ions are studied. In the metal-free systems nucleotide-polyamine molecular complexes have been found to form, in which the interaction centres are the nitrogen atoms of the purine ring N(1) and N(7), oxygen atoms of the phosphate group of the nucleotide (for 3,3,3-tet) and protonated nitrogen atoms of the polyamine. Significant differences in the mode of metallation between the systems with Spm and 3,3,3-tet have been established. In the systems with Spm, the main products are protonated species with [N(7),O] chromophore and the nitrogen N(1) is involved in the intramolecular interaction additionally stabilising the complex. In the systems with 3,3,3-tet the formation of metal-ligand-ligand (MLL) species has been observed, in which the oxygen atoms from the phosphate group and the nitrogen atoms from the polyamine are involved in the metallation, while the N(1) and N(7) atoms from the purine ring of the nucleotide remain outside the inner coordination sphere of the copper ion. The main centre of metallation in the nucleotide, both with Spm and 3,3,3-tet, is the phosphate group of the nucleotide.

  11. Enzyme-free and label-free ultrasensitive electrochemical detection of DNA and adenosine triphosphate by dendritic DNA concatamer-based signal amplification.

    Science.gov (United States)

    Liu, Shufeng; Lin, Ying; Liu, Tao; Cheng, Chuanbin; Wei, Wenji; Wang, Li; Li, Feng

    2014-06-15

    Hybridization chain reaction (HCR) strategy has been well developed for the fabrication of various biosensing platforms for signal amplification. Herein, a novel enzyme-free and label-free ultrasensitive electrochemical DNA biosensing platform for the detection of target DNA and adenosine triphosphate (ATP) was firstly proposed, in which three auxiliary DNA probes were ingeniously designed to construct the dendritic DNA concatamer via HCR strategy and used as hexaammineruthenium(III) chloride (RuHex) carrier for signal amplification. With the developed dendritic DNA concatamer-based signal amplification strategy, the DNA biosensor could achieve an ultrasensitive electrochemical detection of DNA and ATP with a superior detection limit as low as 5 aM and 20 fM, respectively, and also demonstrate a high selectivity for DNA and ATP detection. The currently proposed dendritic DNA concatamer opens a promising direction to construct ultrasensitive DNA biosensing platform for biomolecular detection in bioanalysis and clinical biomedicine, which offers the distinct advantages of simplicity and cost efficiency owing to no need of any kind of enzyme, chemical modification or labeling. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

    Science.gov (United States)

    Zhang, Zhanxia; Sharon, Etery; Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

    2012-06-05

    The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 μM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

  13. Competitive adsorption of uranium(VI) and thorium(IV) ions from aqueous solution using triphosphate-crosslinked magnetic chitosan resins

    International Nuclear Information System (INIS)

    Limin Zhou; Yuyan Jia; Juan Peng; Zhirong Liu; Essam Al-Zaini

    2014-01-01

    The triphosphate-crosslinked magnetic chitosan resins (TPP-MCR) with a diameter range of 200-350 nm were synthesized for the adsorption of U(VI) and Th(IV) ions from aqueous solutions. The adsorption experiments were conducted in both mono-component systems with pure actinide solution and bi-component systems with different U/Th mass ratios. The maximum adsorption capacities in mono-component systems determined by Langmuir model were 169.5 and 146.8 mg g -1 for U(VI) and Th(IV), respectively. In bi-component systems, U(VI) and Th(IV) adsorption capacities were reduced significantly, and the combined sorption capacities were substantially lower (almost halved) compared to those obtained by the addition of sorption capacities using mono-component solutions, indicating that U(VI) and Th(IV) compete for the same sorption sites. Adsorption-desorption experiments for five cycles illustrated the feasibility of the repeated use of TPP-MCR for the adsorption of U(VI) and Th(IV) ions. (author)

  14. Nitrogen doped nanographene structures; study on the adsorption of nucleobases, nucleotides, and their triphosphate derivatives using mixed docking, MD, and QM/MM approaches.

    Science.gov (United States)

    Ghadari, Rahim

    2017-01-28

    The interactions of the nucleobases, nucleotides, and their triphosphate derivatives in both neutral and anionic forms with the nitrogen doped graphenes (NG) were studied using docking and molecular dynamic simulation methods. In docking studies, based on binding energy results, the anionic species and nucleobases were showing the most and the least tendency toward the surface of the NG, respectively. The molecular mechanic/Poisson-Boltzmann surface area results revealed similar results, except for the anionic species; in these studies, the anionic species showed a lesser affinity toward the NG. The time-dependent density functional theory studies were carried out to investigate the effects of the NG on the electronic nature of the investigated ligands; a red-shift in all of the cases was observed. The results of binding energy decomposition and atoms in molecules studies showed that the interactions are van der Waals in nature. The graphitic, pyridinic, and pyrrolic nitrogen atoms which were considered in this study behaved similar to each other.

  15. Di- and Triphosphate Recognition and Sensing with Mono- and Dinuclear Fluorescent Zinc(II) Complexes: Clues for the Design of Selective Chemosensors for Anions in Aqueous Media.

    Science.gov (United States)

    Bartoli, Francesco; Bencini, Andrea; Garau, Alessandra; Giorgi, Claudia; Lippolis, Vito; Lunghi, Alessandro; Totti, Federico; Valtancoli, Barbara

    2016-10-10

    The synthesis of a new ligand (L1) containing two 1,4,7-triazacyclononane ([9]aneN 3 ) moieties linked by a 4,5-dimethylenacridine unit is reported. The binding and fluorescence sensing properties toward Cu 2+ , Zn 2+ , Cd 2+ , and Pb 2+ of L1 and receptor L2, composed of two [9]aneN 3 macrocycles bridged by a 6,6''-dimethylen-2,2':6',2''-terpyridine unit, have been studied by coupling potentiometric, UV/Vis absorption, and emission measurements in aqueous media. Both receptors can selectively detect Zn 2+ thanks to fluorescence emission enhancement upon metal binding. The analysis of the binding and sensing properties of the Zn 2+ complexes toward inorganic anions revealed that the dinuclear Zn 2+ complex of L1 selectively binds and senses the triphosphate anion (TP), whereas the mononuclear Zn 2+ complex of L2 displays selective recognition of diphosphate (DP). Binding of TP or DP induces emission quenching of the Zn 2+ complexes with L1 and L2, respectively. These results are exploited to discuss the role played by pH, number of coordinated metal cations, and binding ability of the bridging units in metal and/or anion coordination and sensing. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Characterization of the Deoxynucleotide Triphosphate Triphosphohydrolase (dNTPase) Activity of the EF1143 Protein from Enterococcus faecalis and Crystal Structure of the Activator-Substrate Complex

    Energy Technology Data Exchange (ETDEWEB)

    Vorontsov, Ivan I.; Minasov, George; Kiryukhina, Olga; Brunzelle, Joseph S.; Shuvalova, Ludmilla; Anderson, Wayne F. (NWU)

    2012-06-19

    The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn{sup 2+}. In contrast, with Mg{sup 2+} hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the {alpha}-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the {alpha}3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.

  17. Coamplification in tumors of KRAS2, type 2 inositol 1,4,5 triphosphate receptor gene, and a novel human gene, KRAG

    Energy Technology Data Exchange (ETDEWEB)

    Heighway, J.; Betticher, D.C.; Altermatt, H.J. [Univ. Hospital of Berne (Switzerland)] [and others

    1996-07-01

    Analysis of a region of DNA, coamplified in tumors with KRAS2, resulted in the identification of the human homologue of the mouse KRAG gene. The gene was widely expressed in range of cell lines, tumors, and normal tissue and demonstrated a high degree of alternate splicing. A human KRAG cDNA sequence, with a structure similar to that encoded by the amplified gene in mouse Y1 adrenal carcinoma cells, was isolated by RT-PCR. The predicted amino acid similarity between the two sequences was 91%, and hydrophobicity plots suggested a structure closely resembling that of transmembrane 4 superfamily members. Identification of a PCR-based restriction fragment length polymorphism allele-specific splicing differences in tumors. Northern analysis of mRNA derived from a range of tissues suggested high level expression in muscle and confirmed alternate splicing. To facilitate the analysis of exon junctions, a YAC clone encoding the genomic sequence was identified. This allowed the localization of KRAG to human chromosome 12p11.2. Isolation of one end of this nonchimeric clone demonstrated a perfect match with a 247-bp sequence within the 3{prime} untranslated region of the type 2 1,4,5-inositol triphosphate receptor gene. Multiplex PCR confirmed the inclusion of both genes. Multiplex PCR confirmed the inclusion of both genes in the KRAS2 amplicon in human malignancy, suggesting that either may contribute to the malignant phenotypes. 35 refs., 6 figs., 1 tab.

  18. Mechanism of action of minoxidil in the treatment of androgenetic alopecia is likely mediated by mitochondrial adenosine triphosphate synthase-induced stem cell differentiation.

    Science.gov (United States)

    Goren, A; Naccarato, T; Situm, M; Kovacevic, M; Lotti, T; McCoy, J

    2017-01-01

    Topical minoxidil is the only topical drug approved by the US Food and Drug Administration (FDA) for the treatment of androgenetic alopecia. However, the exact mechanism by which minoxidil stimulates anagen phase and promotes hair growth is not fully understood. In the late telegen phase of the hair follicle growth cycle, stem cells located in the bulge region differentiate and re-enter anagen phase, a period of growth lasting 2-6 years. In androgenetic alopecia, the anagen phase is shortened and a progressive miniaturization of hair follicles occurs, eventually leading to hair loss. Several studies have demonstrated that minoxidil increases the amount of intracellular Ca2+, which has been shown to up-regulate the enzyme adenosine triphosphate (ATP) synthase. A recent study demonstrated that ATP synthase, independent of its role in ATP synthesis, promotes stem cell differentiation. As such, we propose that minoxidil induced Ca2+ influx can increase stem cell differentiation and may be a key factor in the mechanism by which minoxidil facilitates hair growth. Based on our theory, we provide a roadmap for the development of a new class of drugs for the treatment of androgenetic alopecia.

  19. Adenosine Triphosphate-sensitive Micro-reentrant Atrial Tachycardia Originating from the Crista Terminalis in a Patient with Chronic Renal Failure due to Thrombotic Thrombocytopenic Purpura

    Directory of Open Access Journals (Sweden)

    Shinya Sugiura, MD

    2009-01-01

    Full Text Available A 57-year-old woman with chronic renal failure due to the thrombotic thrombocytopenic purpura complained of palpitation. A 12-lead ECG showed supraventricular tachycardia with a cycle length of 375 ms. During the electrophysiological study, a tachycardia with a cycle length of 375 ms was reproducibly induced and terminated by atrial extrastimulation. The tachycardia exhibited an inverse relationship between the coupling interval of extrastimulus initiating the tachycardia, and the first postpacing return cycle, as well as an increasing pattern of resetting the tachycardia with an atrial extrastimulus. Ventricular burst pacing during tachycardia produced AV dissociation. Intravenous injections of a low dose (4 mg of adenosine triphosphate (ATP terminated the tachycardia without a preceding atrio-His bundle block. The tachycardia was diagnosed as an ATP-sensitive micro-reentrant atrial tachycardia. Real-time endocardial activation mapping using an electroanatomical mapping system revealed that the earliest activation site of the tachycardia was located at the midlateral portion of the crista terminalis. The tachycardia was abolished by focal ablation targeting the earliest activation site during tachycardia. This is the first reported case of an ATP-sensitive micro-reentrant atrial tachycardia associated with thrombotic thrombocytopenic purpura.

  20. Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

    International Nuclear Information System (INIS)

    Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

    1987-01-01

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

  1. Increased tolerance to stress in cardiac expressed gain-of-function of adenosine triphosphate-sensitive potassium channel subunit Kir6.1.

    Science.gov (United States)

    Henn, Matthew C; Janjua, M Burhan; Zhang, Haixia; Kanter, Evelyn M; Makepeace, Carol M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2016-12-01

    The adenosine triphosphate-sensitive potassium (K ATP ) channel opener diazoxide (DZX) prevents myocyte volume derangement and reduced contractility secondary to stress. K ATP channels are composed of pore-forming (Kir6.1 or Kir6.2) and regulatory (sulfonylurea receptor, SUR1 or SUR2) subunits. Gain of function (GOF) of Kir6.1 subunits has been implicated in cardiac pathology in Cantu syndrome in humans (cardiomegaly, lymphedema, and pericardial effusions). We hypothesized that GOF of Kir6.1 subunits would result in altered myocyte response to stress. Isolated cardiac myocytes from wild type (WT) and transgenic Kir6.1GOF mice were exposed to Tyrode's physiologic solution for 20 min, test solution (Tyrode's or stress [hyperkalemic cardioplegia {CPG, known myocyte stress}] +/- K ATP channel opener DZX), followed by Tyrode's for 20 min. Myocyte volume and contractility were measured and compared. WT myocytes demonstrated significant swelling in response to stress, but significantly less swelling was seen in Kir6.1GOF myocytes. DZX prevented swelling secondary to CPG in WT but resulted in a nonsignificant reduction in swelling in Kir6.1GOF myocytes. Both WT and Kir6.1GOF myocytes demonstrated a reduction in contractility during stress, although this was only significant in Kir6.1GOF myocytes. DZX was not associated with an improvement in contractility in Kir6.1GOF myocytes following stress. Similar to previous results in Kir6.1(-/-) myocytes, Kir6.1GOF myocytes demonstrate resistance (less volume derangement) to stress of cardioplegia. Understanding the role of Kir6.1 in myocyte response to stress may aid in the treatment of patients with Cantu syndrome and warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Artificial oxygen carrier with pharmacologic actions of adenosine-5'-triphosphate, adenosine, and reduced glutathione formulated to treat an array of medical conditions.

    Science.gov (United States)

    Simoni, Jan; Simoni, Grace; Moeller, John F; Feola, Mario; Wesson, Donald E

    2014-08-01

    Effective artificial oxygen carriers may offer a solution to tackling current transfusion medicine challenges such as blood shortages, red blood cell storage lesions, and transmission of emerging pathogens. These products, could provide additional therapeutic benefits besides oxygen delivery for an array of medical conditions. To meet these needs, we developed a hemoglobin (Hb)-based oxygen carrier, HemoTech, which utilizes the concept of pharmacologic cross-linking. It consists of purified bovine Hb cross-linked intramolecularly with open ring adenosine-5'-triphosphate (ATP) and intermolecularly with open ring adenosine, and conjugated with reduced glutathione (GSH). In this composition, ATP prevents Hb dimerization, and adenosine promotes formation of Hb polymers as well as counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine receptors. ATP also serves as a regulator of vascular tone through activation of purinergic receptors. GSH blocks Hb's extravasation and glomerular filtration by lowering the isoelectric point, as well as shields heme from nitric oxide and reactive oxygen species. HemoTech and its manufacturing technology have been broadly tested, including viral and prion clearance validation studies and various nonclinical pharmacology, toxicology, genotoxicity, and efficacy tests. The clinical proof-of-concept was carried out in sickle cell anemia subjects. The preclinical and clinical studies indicate that HemoTech works as a physiologic oxygen carrier and has efficacy in treating: (i) acute blood loss anemia by providing a temporary oxygen bridge while stimulating an endogenous erythropoietic response; (ii) sickle cell disease by counteracting vaso-occlusive/inflammatory episodes and anemia; and (iii) ischemic vascular diseases particularly thrombotic and restenotic events. The pharmacologic cross-linking of Hb with ATP, adenosine, and GSH showed usefulness in designing an artificial oxygen carrier for

  3. Sensitive determination of adenosine disodium triphosphate in soil, milk, and pharmaceutical formulation by enoxacin–europium (III) fluorescence complex in solution

    International Nuclear Information System (INIS)

    Alam, Al-Mahmnur; Kamruzzaman, Mohammad; Hak Lee, Sang; Ho Kim, Young; Jin Jo, Hae; Hong Kim, Sung; Park, Sang-Ryoul

    2012-01-01

    A new spectroflurometric method for the determination of adenosine disodium triphosphate (ATP) is developed. Fluorometric interaction between ATP and enoxacin (ENX)–Eu 3+ complex was studied using UV–vis and fluorescence spectroscopy. Weak luminescence spectra of Eu 3+ were enhanced after complexation with ENX at 589 nm and 614 nm upon excitation at 395 nm due to energy transfer from the ligand to the lanthanide ion. It was observed that luminescence spectrum of Eu 3+ was strongly enhanced further at 614 nm after incorporation of ATP into the ENX–Eu 3+ complex. Under optimal conditions, the enhancement of luminescence at 614 nm was responded linearly with the concentration of ATP. The linearity was maintained in the range of 1.5×10 −10 –1.15×10 −8 M (R=0.9973) with the limit of detection (3σ) of 4.71×10 −11 M. The relative standard deviation (RSD) for 9 repeated measurements of 1×10 −9 M ATP was 1.25%. Successful determinations of ATP in soil, milk, and a pharmaceutical formulation with the proposed method were demonstrated. - Highlights: ► Weak luminescence of Eu 3+ was enhanced at 614 nm after formation of complex with ENX. ► Energy transfer occurs through FRET from ENX to Eu 3+ upon excitation. ► Luminescence signal was further enhanced when ATP conjugates with ENX–Eu 3+ complex. ► Luminescence intensity of Eu 3+ at 614 nm was correlated with concentration of ATP. ► The method was applied to determine ATP in soil, milk, and pharmaceutical samples.

  4. Determination of nucleoside analog mono-, di-, and tri-phosphates in cellular matrix by solid phase extraction and ultra-sensitive LC-MS/MS detection

    Science.gov (United States)

    Bushman, Lane R.; Kiser, Jennifer J.; Rower, Joseph E.; Klein, Brandon; Zheng, Jia-Hua; Ray, Michelle L.; Anderson, Peter L.

    2011-01-01

    An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleoside analog MP-, DP-, and TP- determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.50 to 2000 fmol/sample, and that for 3TC/FTC was 0.10 to 200 pmol/sample. Nucleoside analog MP-, DP-, and TP- determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.10 to 100 pmol/sample, and 5.00 to 2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research. PMID:21715120

  5. Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours.

    Directory of Open Access Journals (Sweden)

    Yiying Cai

    Full Text Available Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.100 strains of carbapenem-resistant (CR GNB [30 Acinetobacter baumannii (AB, 30 Pseudomonas aeruginosa (PA and 40 Klebsiella pneumoniae (KP] were used. Bacterial suspensions (105 CFU/ml were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (TRLU to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA [(sensitivity + specificity/2] of TRLU values was determined. External validation was further done using 50 additional CR-GNB.Predictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, UA = 89%. When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (TRLU = 0.81, UA = 91%, and lowest for AB (TRLU = 0.83, UA = 87%. Upon external validation, high overall accuracy (91% was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.

  6. Monitoring of intracellular adenosine triphosphate in CD4(+) T cells to predict the occurrence of cytomegalovirus disease in kidney transplant recipients.

    Science.gov (United States)

    Pérez-Jacoiste Asín, María Asunción; Fernández-Ruiz, Mario; López-Medrano, Francisco; Aquilino, Carolina; González, Esther; Ruiz-Merlo, Tamara; Gutiérrez, Eduardo; San Juan, Rafael; Paz-Artal, Estela; Andrés, Amado; Aguado, José Maria

    2016-10-01

    The measurement of intracellular concentrations of adenosine triphosphate (iATP) in phytohemagglutinin-stimulated CD4(+) T cells constitutes a surrogate marker for post-transplant cell-mediated immunity (CMI). This assay has shown suboptimal accuracy for predicting infection after kidney transplantation (KT). We hypothesize that its predictive capacity depends on the specific contribution of the CMI to host-pathogen interactions. We assessed iATP levels in 100 KT recipients at baseline and months 1, 3, and 6 (363 measurements). No association was found between iATP at month 1 and the risk for overall or bacterial infection, although such association was evident for cytomegalovirus (CMV) disease (multivariate-adjusted hazard ratio [per 50-unit increment]: 0.83; P-value = 0.048). There were no significant differences in mean iATP between stable patients (319.4 ng/ml) and those developing overall (304.1 ng/ml) or bacterial infection (346.9 ng/ml) over the 45 days following monitoring. However, iATP was significantly lower in patients who developed CMV disease (223.5 ng/ml; P-values <0.002). The optimal cutoff (265 ng/ml) for predicting CMV disease in patients not receiving antiviral prophylaxis yielded sensitivity, specificity, positive, and negative predictive values of 85.7%, 68.3%, 15.2%, and 98.6%, respectively. In conclusion, a non-pathogen-specific monitoring of CMI by means of iATP informs the risk of CMV disease in KT recipients. © 2016 Steunstichting ESOT.

  7. Deciphering common recognition principles of nucleoside mono/di and tri-phosphates binding in diverse proteins via structural matching of their binding sites.

    Science.gov (United States)

    Bhagavat, Raghu; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-09-01

    Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Adenosine 5′-triphosphate (ATP supplements are not orally bioavailable: a randomized, placebo-controlled cross-over trial in healthy humans

    Directory of Open Access Journals (Sweden)

    Arts Ilja CW

    2012-04-01

    Full Text Available Abstract Background Nutritional supplements designed to increase adenosine 5′-triphosphate (ATP concentrations are commonly used by athletes as ergogenic aids. ATP is the primary source of energy for the cells, and supplementation may enhance the ability to maintain high ATP turnover during high-intensity exercise. Oral ATP supplements have beneficial effects in some but not all studies examining physical performance. One of the remaining questions is whether orally administered ATP is bioavailable. We investigated whether acute supplementation with oral ATP administered as enteric-coated pellets led to increased concentrations of ATP or its metabolites in the circulation. Methods Eight healthy volunteers participated in a cross-over study. Participants were given in random order single doses of 5000 mg ATP or placebo. To prevent degradation of ATP in the acidic environment of the stomach, the supplement was administered via two types of pH-sensitive, enteric-coated pellets (targeted at release in the proximal or distal small intestine, or via a naso-duodenal tube. Blood ATP and metabolite concentrations were monitored by HPLC for 4.5 h (naso-duodenal tube or 7 h (pellets post-administration. Areas under the concentration vs. time curve were calculated and compared by paired-samples t-tests. Results ATP concentrations in blood did not increase after ATP supplementation via enteric-coated pellets or naso-duodenal tube. In contrast, concentrations of the final catabolic product of ATP, uric acid, were significantly increased compared to placebo by ~50% after administration via proximal-release pellets (P = 0.003 and naso-duodenal tube (P = 0.001, but not after administration via distal-release pellets. Conclusions A single dose of orally administered ATP is not bioavailable, and this may explain why several studies did not find ergogenic effects of oral ATP supplementation. On the other hand, increases in uric acid after release of

  9. Quantitative circumferential strain analysis using adenosine triphosphate-stress/rest 3-T tagged magnetic resonance to evaluate regional contractile dysfunction in ischemic heart disease.

    Science.gov (United States)

    Nakamura, Masashi; Kido, Tomoyuki; Kido, Teruhito; Tanabe, Yuki; Matsuda, Takuya; Nishiyama, Yoshiko; Miyagawa, Masao; Mochizuki, Teruhito

    2015-08-01

    We evaluated whether a quantitative circumferential strain (CS) analysis using adenosine triphosphate (ATP)-stress/rest 3-T tagged magnetic resonance (MR) imaging can depict myocardial ischemia as contractile dysfunction during stress in patients with suspected coronary artery disease (CAD). We evaluated whether it can differentiate between non-ischemia, myocardial ischemia, and infarction. We assessed its diagnostic performance in comparison with ATP-stress myocardial perfusion MR and late gadolinium enhancement (LGE)-MR imaging. In 38 patients suspected of having CAD, myocardial segments were categorized as non-ischemic (n=485), ischemic (n=74), or infarcted (n=49) from the results of perfusion MR and LGE-MR. The peak negative CS value, peak circumferential systolic strain rate (CSR), and time-to-peak CS were measured in 16 segments. A cutoff value of -12.0% for CS at rest allowed differentiation between infarcted and other segments with a sensitivity of 79%, specificity of 76%, accuracy of 76%, and an area under the curve (AUC) of 0.81. Additionally, a cutoff value of 477.3ms for time-to-peak CS at rest allowed differentiation between infarcted and other segments with a sensitivity of 61%, specificity of 91%, accuracy of 88%, and an AUC of 0.75. The differences in CS values between ATP-stress and rest conditions (ΔCS) in non-ischemic segments (median [first quartile, third quartile] -1.7 [-3.2, -0.1] %) were smaller than in segments with ischemia (+1.1 [+0.3, +2.3] %, p<0.001). A cutoff value of +0.3% for the ΔCS value could differentiate segments with ischemia from non-ischemic segments with a sensitivity of 75%, a specificity of 82%, an accuracy of 82%, and an AUC of 0.86. Circumferential strain analysis using tagged MR can quantitatively assess contractile dysfunction in ischemic and infarcted myocardium. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Quantitative circumferential strain analysis using adenosine triphosphate-stress/rest 3-T tagged magnetic resonance to evaluate regional contractile dysfunction in ischemic heart disease

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Masashi, E-mail: m.nakamura1230@gmail.com [Department of Radiology, Ehime University Graduate School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295 (Japan); Kido, Tomoyuki [Department of Radiology, Saiseikai Matsuyama Hospital, Ehime 791-0295 (Japan); Kido, Teruhito; Tanabe, Yuki; Matsuda, Takuya; Nishiyama, Yoshiko; Miyagawa, Masao; Mochizuki, Teruhito [Department of Radiology, Ehime University Graduate School of Medicine, Shitsukawa, Toon-city, Ehime 791-0295 (Japan)

    2015-08-15

    Highlights: • Infarcted segments could be differentiated from non-ischemic and ischemic segments with high sensitivity and specificity under at rest conditions. • The time-to-peak circumferential strain values in infarcted segments were more significantly delayed than those in non-ischemic and ischemic segments. • Both circumferential strain and circumferential systolic strain rate values under ATP-stress conditions were significantly lower in ischemic segments than in non-ischemic segments. • Subtracting stress and rest circumferential strain had a higher diagnostic capability for ischemia relative to only utilizing rest or ATP-stress circumferential strain values. • A circumferential strain analysis using tagged MR can quantitatively assess contractile dysfunction in ischemic and infarcted myocardium. - Abstract: Purpose: We evaluated whether a quantitative circumferential strain (CS) analysis using adenosine triphosphate (ATP)-stress/rest 3-T tagged magnetic resonance (MR) imaging can depict myocardial ischemia as contractile dysfunction during stress in patients with suspected coronary artery disease (CAD). We evaluated whether it can differentiate between non-ischemia, myocardial ischemia, and infarction. We assessed its diagnostic performance in comparison with ATP-stress myocardial perfusion MR and late gadolinium enhancement (LGE)-MR imaging. Methods: In 38 patients suspected of having CAD, myocardial segments were categorized as non-ischemic (n = 485), ischemic (n = 74), or infarcted (n = 49) from the results of perfusion MR and LGE-MR. The peak negative CS value, peak circumferential systolic strain rate (CSR), and time-to-peak CS were measured in 16 segments. Results: A cutoff value of −12.0% for CS at rest allowed differentiation between infarcted and other segments with a sensitivity of 79%, specificity of 76%, accuracy of 76%, and an area under the curve (AUC) of 0.81. Additionally, a cutoff value of 477.3 ms for time-to-peak CS at rest

  11. The P2X7 receptor antagonist, oxidized adenosine triphosphate, ameliorates renal ischemia-reperfusion injury by expansion of regulatory T cells.

    Science.gov (United States)

    Koo, Tai Yeon; Lee, Jae-Ghi; Yan, Ji-Jing; Jang, Joon Young; Ju, Kyung Don; Han, Miyeun; Oh, Kook-Hwan; Ahn, Curie; Yang, Jaeseok

    2017-08-01

    Extracellular adenosine triphosphate (ATP) binds to purinergic receptors and, as a danger molecule, promotes inflammatory responses. Here we tested whether periodate-oxidized ATP (oATP), a P2X7 receptor (P2X7R) antagonist can attenuate renal ischemia-reperfusion injury and clarify the related cellular mechanisms. Treatment with oATP prior to ischemia-reperfusion injury decreased blood urea nitrogen, serum creatinine, the tubular injury score, and tubular epithelial cell apoptosis after injury. The infiltration of dendritic cells, neutrophils, macrophages, CD69 + CD4 + , and CD44 + CD4 + T cells was attenuated, but renal Foxp3 + CD4 + Treg infiltration was increased by oATP. The levels of IL-6 and CCL2 were reduced in the oATP group. Additionally, oATP treatment following injury improved renal function, decreased the infiltration of innate and adaptive effector cells, and increased the renal infiltration of Foxp3 + CD4 + Tregs. Post-ischemia-reperfusion injury oATP treatment increased tubular cell proliferation and reduced renal fibrosis. oATP treatment attenuated renal functional deterioration after ischemia-reperfusion injury in RAG-1 knockout mice; however, Treg depletion using PC61 abrogated the beneficial effects of oATP in wild-type mice. Furthermore, oATP treatment after transfer of Tregs from wild-type mice improved the beneficial effects of Tregs on ischemia-reperfusion injury, but treatment after transfer of Tregs from P2X7R knockout mice did not. Renal ischemia-reperfusion injury was also attenuated in P2X7R knockout mice. Experiments using bone marrow chimeras established that P2X7R expression on hematopoietic cells rather than non-hematopoietic cells, such as tubular epithelial cells, plays a major role in ischemia-reperfusion injury. Thus, oATP attenuated acute renal damage and facilitated renal recovery in ischemia-reperfusion injury by expansion of Tregs. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights

  12. Prevention of isoflurane-induced preconditioning by 5-hydroxydecanoate and gadolinium: possible involvement of mitochondrial adenosine triphosphate-sensitive potassium and stretch-activated channels.

    Science.gov (United States)

    Piriou, V; Chiari, P; Knezynski, S; Bastien, O; Loufoua, J; Lehot, J J; Foëx, P; Annat, G; Ovize, M

    2000-09-01

    Both mitochondrial adenosine triphosphate-sensitive potassium (MKATP) channels (selectively blocked by 5-hydroxydecanoate) and stretch-activated channels (blocked by gadolinium) have been involved in the mechanism of ischemic preconditioning. Isoflurane can reproduce the protection afforded by ischemic preconditioning. We sought to determine whether isoflurane-induced preconditioning may involve MKATP and stretch-activated channels. Anesthetized open-chest rabbits underwent 30 min of coronary occlusion followed by 3 h of reperfusion. Before this, rabbits were randomized into one of six groups and underwent a treatment period consisting of either no intervention for 40 min (control group; n = 9) or 15 min of isoflurane inhalation (1.1% end tidal) followed by a 15-min washout period (isoflurane group; n = 9). The two groups received an intravenous bolus dose of either 5-hydroxydecanoate (5 mg/kg) or gadolinium (40 micromol/kg) before coronary occlusion and reperfusion (5-hydroxydecanoate, n = 9; gadolinium, n = 7). Two additional groups received 5-hydroxydecanoate or gadolinium before isoflurane exposure (isoflurane-5-hydroxydecanoate, n = 10; isoflurane-gadolinium, n = 8). Area at risk and infarct size were assessed by blue dye injection and tetrazolium chloride staining. Area at risk was comparable among the six groups (29 +/- 7, 30 +/- 5, 27 +/- 6, 35 +/- 7, 31 +/- 7, and 27 +/- 4% of the left ventricle in the control, isoflurane, isoflurane-5-hydroxydecanoate, 5-hydroxydecanoate, isoflurane-gadolinium, and gadolinium groups, respectively). Infarct size averaged 60 +/- 20% (SD) in untreated controls versus 54 +/- 27 and 65 +/- 15% of the risk zone in 5-hydroxydecanoate- and gadolinium-treated controls (P = nonsignificant). In contrast, infarct size in the isoflurane group was significantly reduced to 26 +/- 11% of the risk zone (P < 0.05 vs.control). Both 5-hydroxydecanoate and gadolinium prevented this attenuation: infarct size averaged 68 +/- 23 and 56 +/- 21

  13. Cross-coupling reactions of unprotected halopurine bases, nucleosides, nucleotides and nucleoside triphosphates with 4-boronophenylalanine in water. Synthesis of (purin-8-yl)- and (purin-6-yl)phenylalanines.

    Science.gov (United States)

    Capek, Petr; Pohl, Radek; Hocek, Michal

    2006-06-07

    An expeditious and highly efficient single-step methodology for the introduction of a phenylalanine moiety into position 8 and 6 of the purine scaffold was developed based on aqueous-phase Pd-catalysed Suzuki-Miyaura cross-coupling reactions of unprotected 4-boronophenylalanine with 8-bromo- or 6-chloropurines. The scope of the methodology was demonstrated by syntheses of unprotected (adenin-8-yl)phenylalanine base, nucleosides, nucleotides and nucleoside triphosphates as well as (purin-6-yl)phenylalanine base and nucleosides. All these products were obtained in high yields and in optically pure form.

  14. Presynaptic alpha2-adrenoceptor-mediated modulation of adenosine 5' triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein.

    Science.gov (United States)

    Bobalova, J; Mutafova-Yambolieva, V N

    2001-02-01

    1. The modulatory effects of agonists and antagonists of prejunctional alpha2-adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5' triphosphate (ATP) were measured in endothelium-denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long-duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2. The EFS-evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 microM) and guanethidine (10 microM) in the artery and vein. Desipramine (10 microM), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)-evoked overflow of NA in both artery and vein. EFS-evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS-evoked overflow of ATP was equal in the artery and vein. 3. Stimulation of alpha2-adrenoceptors with clonidine (0.1 microM) and oxymethazoline (0.3 microM) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the alpha2-adrenoceptor agonists in the artery and vein. 4. In artery, blockade of alpha2-adrenoceptors with yohimbine at a concentration of 0.1 microM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 microM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 microM) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 microM) increased the NA overflow in artery only at 4 Hz, whereas in vein idazoxan

  15. Comparison of the Immunomagnetic Separation/Adenosine Triphosphate Rapid Method and the Modified mTEC Membrane-Filtration Method for Enumeration of Escherichia coli

    Science.gov (United States)

    Brady, Amie M.G.; Bushon, Rebecca N.; Bertke, Erin E.

    2009-01-01

    Water quality at beaches is monitored for fecal indicator bacteria by traditional, culture-based methods that can take 18 to 24 hours to obtain results. A rapid detection method that provides estimated concentrations of fecal indicator bacteria within 1 hour from the start of sample processing would allow beach managers to post advisories or close the beach when the conditions are actually considered unsafe instead of a day later, when conditions may have changed. A rapid method that couples immunomagnetic separation with adenosine triphosphate detection (IMS/ATP rapid method) was evaluated through monitoring of Escherichia coli (E. coli) at three Lake Erie beaches in Ohio (Edgewater and Villa Angela in Cleveland and Huntington in Bay Village). Beach water samples were collected between 4 and 5 days per week during the recreational seasons (May through September) of 2006 and 2007. Composite samples were created in the lab from two point samples collected at each beach and were shown to be comparable substitutes for analysis of two individual samples. E. coli concentrations in composite samples, as determined by the culture-based method, ranged from 4 to 24,000 colony-forming units per 100 milliliters during this study across all beaches. Turbidity also was measured for each sample and ranged from 0.8 to 260 neophelometric turbidity ratio units. Environmental variables were noted at the time of sampling, including number of birds at the beach and wave height. Rainfall amounts were measured at National Weather Service stations at local airports. Turbidity, rainfall, and wave height were significantly related to the culture-based method results each year and for both years combined at each beach. The number of birds at the beach was significantly related to the culture-based method results only at Edgewater during 2006 and during both years combined. Results of the IMS/ATP method were compared to results of the culture-based method for samples by year for each beach

  16. Interactions of the products, 8-oxo-dGMP, dGMP, and pyrophosphate with the MutT nucleoside triphosphate pyrophosphohydrolase.

    Science.gov (United States)

    Saraswat, Vibhor; Massiah, Michael A; Lopez, Gregory; Amzel, L Mario; Mildvan, Albert S

    2002-12-31

    The MutT enzyme from E. coli, in the presence of a divalent cation, catalyzes the hydrolysis of nucleoside- and deoxynucleoside-triphosphate (NTP) substrates by nucleophilic substitution at Pbeta, to yield a nucleotide (NMP) and PPi. The best substrate of MutT is believed to be the mutagenic nucleotide 8-oxo-dGTP, on the basis of its 10(3.4)-fold lower K(m) than that of dGTP (Maki, H., and Sekiguchi, M. (1992) Nature 355, 273-275). To determine the true affinity of MutT for an 8-oxo-nucleotide and to elucidate the kinetic scheme, product inhibition by 8-oxo-dGMP and dGMP and direct binding of these nucleotides to MutT were studied. With Mg(2+)-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitive inhibitor with K(I)(sl)(o)(pe) = 49 nM, which is 10(4.6)-fold lower than the K(I)(sl)(o)(pe)of dGMP (1.7 mM). Similarly, the K(I)(intercept) of 8-oxo-dGMP is 10(4.0)-fold lower than that of dGMP. PPi is a linear uncompetitive inhibitor, suggesting that it dissociates first from the product complex, followed by the nucleotide. Noncompetitive inhibition by dGMP and 8-oxo-dGMP indicates an "iso" mechanism in which the nucleotide product leaves an altered form of the enzyme which slowly reverts to the form which binds substrate. Consistent with this kinetic scheme, (1)H-(15)N HSQC titration of MutT with dGMP reveals weak binding and fast exchange from one site with a K(D) = 1.8 mM, in agreement with its K(I)(sl)(o)(pe). With 8-oxo-dGMP, tight binding and slow exchange (n = 1.0 +/- 0.1, K(D) MutT with 8-oxo-dGMP yields a K(D) of 52 nM, in agreement with its K(I)(sl)(o)(pe). Changing the metal activator from Mg(2+) to Mn(2+) had little effect on the K(I)(sl)(o)(pe) of dGMP or of 8-oxo-dGMP, consistent with the second-sphere enzyme-M(2+)-H(2)O-NTP-M(2+) complex found by NMR (Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211), but it decreased the K(I) of PPi 12-fold, suggesting direct coordination of the PPi

  17. Formation of Sodium cyclo-Triphosphate by the Thermal Reaction of Sodium Dihydrogenphosphate-Organic Nitrogen Compound Mixtures; Rinsan nisuiso natoriumu-yuki chisso kagobutsu kongokei no kanetsu ni yoru cyclo-sanrinsan natoriumu no seisei

    Energy Technology Data Exchange (ETDEWEB)

    Takenaka, Atsushi.; Kikui, Satoshi. [Yonago National College of Technology, Tottori (Japan). Department of Materials Science; Motooka, Itaru.; Nariai, Hiroyuki. [Kobe University, Hyogo (Japan). Department of Chemical Science and Engineering

    1998-11-10

    Mixtures of sodium dihydrogenphosphate (NaH{sub 2}PO{sub 4}) with biuret, cyanuric acid, or melamine were heated in a stream of nitrogen gas. Crystalline sodium polyphosphate of high-temperature type of Maddrell`s salt was produced at above 300 degree C in the NaH{sub 2}PO{sub 4}-cyanuric acid or NaH{sub 2}PO{sub 4}-melamine mixtures. Sodium cyclo-trihosphate was produced in the NaH{sub 2}PO{sub 3}-biuret mixture in a similar manner to the thermal reaction of NaH{sub 2}PO{sub 4}-urea mixture reported previously, which was considered to be responsible for the enhanced water-vapor pressure in samples caused by the the formation of a kind of (shell.) An increase in amount of biuret added was available for the formation of cyclo-triphosphate. (author)

  18. Formation of Sodium cyclo-Triphosphate by the Thermal Reaction of Sodium Dihydrogenphosphate-Organic Nitrogen Compound Mixtures. Rinsan nisuiso natoriumu-yuki chisso kagobutsu kongokei no kanetsu ni yoru cyclo-sanrinsan natoriumu no seisei

    Energy Technology Data Exchange (ETDEWEB)

    Takenaka, Atsushi.; Kikui, Satoshi. (Yonago National College of Technology, Tottori (Japan). Department of Materials Science); Motooka, Itaru.; Nariai, Hiroyuki. (Kobe University, Hyogo (Japan). Department of Chemical Science and Engineering)

    1998-11-10

    Mixtures of sodium dihydrogenphosphate (NaH[sub 2]PO[sub 4]) with biuret, cyanuric acid, or melamine were heated in a stream of nitrogen gas. Crystalline sodium polyphosphate of high-temperature type of Maddrell's salt was produced at above 300 degree C in the NaH[sub 2]PO[sub 4]-cyanuric acid or NaH[sub 2]PO[sub 4]-melamine mixtures. Sodium cyclo-trihosphate was produced in the NaH[sub 2]PO[sub 3]-biuret mixture in a similar manner to the thermal reaction of NaH[sub 2]PO[sub 4]-urea mixture reported previously, which was considered to be responsible for the enhanced water-vapor pressure in samples caused by the the formation of a kind of (shell.) An increase in amount of biuret added was available for the formation of cyclo-triphosphate. (author)

  19. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); Scientific Opinion on the substantiation of health claims related to sugar-free chewing gum with pyro- and triphosphates and reduction of calculus formation (ID 1309) pursuant to Article 13(1) of Regulation (EC) No 1924/2006

    DEFF Research Database (Denmark)

    Tetens, Inge

    claims in relation to free chewing gum with pyro- and triphosphates and reduction of calculus formation. The scientific substantiation is based on the information provided by the Member States in the consolidated list of Article 13 health claims and references that EFSA has received from Member States...... calculus/tartar formation, gums health”. The target population is assumed to be the general population. The Panel considers that reduction of calculus formation at sites which are most important for dental health is a beneficial physiological effect. No human studies have been provided from which...... conclusions could be drawn for the scientific substantiation of a claim on the use of sugar-free chewing gum with pyro- and triphosphates and the reduction of calculus formation at sites which are most important for dental health (e.g. gingival margin or between teeth). On the basis of the data presented...

  20. Spectral studies of lanthanide-nucleic acid component interaction: complexes of adenine, adenosine, adenosine 5'-mono-, adenosine 5'-di- and adenosine 5' tri-phosphates with praseodymium(III)

    International Nuclear Information System (INIS)

    Joseph, George; Anjaiah, K.; Misra, S.N.

    1990-01-01

    The interactions of adenine, adenosine, adenosine 5'-mono-, adenosine 5'-di-and adenosine 5'-tri-phosphates with praseodymium(III) have been studied in different stoichiometries and at varying hydrogen ion concentrations by absorption spectral studies. The sharp bands in the spectra have been individually analysed by Gaussian curve analysis, and various spectral parameters have been computed using partial and multiple regression methods on an HP-1000/45 computer. The changes in and the magnitudes of these parameters have been correlated with the degrees of outer- and inner-sphere coordination around praseodymium(III). Crystalline complexes of the type: Pr(nucleotide) 2 (H 2 O) 2 (where nucleotide = AMP, ADP and ATP) have been characterized on the basis of analytical, IR and 1 H NMR spectral data. These studies indicate that the binding of the nucleotide is through phosphoric oxygen. These complexes in aqueous medium show significant ionization which supports the observed weak 4f-4f bands, lower values of nephelauxetic effect and the parameters derived from coulombic and spin-orbit interactions. (author). 3 t abs., 28 refs

  1. Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide.

    Science.gov (United States)

    Das, Ushati; Wang, Li Kai; Smith, Paul; Munir, Annum; Shuman, Stewart

    2014-12-01

    Clostridium thermocellum polynucleotide kinase (CthPnk), the 5'-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5'-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg(2+) and a 5'-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2'-hydroxyls of the 5' terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5'-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg(2+) and a 5'-OH DNA yielded a mixed substrate-product complex with GTP-Mg(2+) and 5'-PO4 DNA, wherein the product 5' phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. (Alton Ochsner Medical Foundation, New Orleans, LA (USA))

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  3. Intracellular Secretory Leukoprotease Inhibitor Modulates Inositol 1,4,5-Triphosphate Generation and Exerts an Anti-Inflammatory Effect on Neutrophils of Individuals with Cystic Fibrosis and Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Emer P. Reeves

    2013-01-01

    Full Text Available Secretory leukoprotease inhibitor (SLPI is an anti-inflammatory protein present in respiratory secretions. Whilst epithelial cell SLPI is extensively studied, neutrophil associated SLPI is poorly characterised. Neutrophil function including chemotaxis and degranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca2+ levels which is mediated by production of inositol 1,4,5-triphosphate (IP3 in response to G-protein-coupled receptor (GPCR stimuli. The aim of this study was to investigate the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease. Neutrophils were isolated from healthy controls (n=10, individuals with cystic fibrosis (CF (n=5 or chronic obstructive pulmonary disease (COPD (n=5. Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP and interleukin(IL-8 induced neutrophil chemotaxis (P<0.05 and decreased degranulation of matrix metalloprotease-9 (MMP-9, hCAP-18, and myeloperoxidase (MPO (P<0.05. The mechanism of inhibition involved modulation of cytosolic IP3 production and downstream Ca2+ flux. The described attenuation of Ca2+ flux was overcome by inclusion of exogenous IP3 in electropermeabilized cells. Inhibition of IP3 generation and Ca2+ flux by SLPI may represent a novel anti-inflammatory mechanism, thus strengthening the attractiveness of SLPI as a potential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF, non-CF bronchiectasis, and COPD.

  4. Covalently linked immunomagnetic separation/adenosine triphosphate technique (Cov-IMS/ATP) enables rapid, in-field detection and quantification of Escherichia coli and Enterococcus spp. in freshwater and marine environments.

    Science.gov (United States)

    Lee, C M; Griffith, J F; Kaiser, W; Jay, J A

    2010-07-01

    Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody-bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems. Covalently linked complexes were 58-89% more robust than antibody-bead complexes used in previous studies. Freshwater and marine water samples analysed using covalently linked immunomagnetic separation/adenosine triphosphate quantification technique (Cov-IMS/ATP) and culture-based methods yielded good correlations for E. coli (R = 0·87) and Enterococcus spp. (R = 0·94), with method detection limits below EPA recreational water quality health standards for single standard exceedances (E. coli- 38 cells per 100 ml; Enterococcus spp. - 25 cells per 100 ml). Cov-IMS/ATP correctly classified 87% of E. coli and 94% of Enterococcus spp. samples based on these water quality standards. Cov-IMS/ATP was also used as a field method to rapidly distinguish differential loading of E. coli between two stream channels to their confluence. Cov-IMS/ATP is a robust, in-field detection method for determining water quality of both fresh and marine water systems as well as differential loading of FIB from two converging channels. To our knowledge, this is the first work to present a viable rapid, in-field assay for measuring FIB concentrations in marine water environments. Cov-IMS/ATP is a potential alternative detection method, particularly in areas with limited laboratory support and resources, because of its increased economy and portability. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  5. 4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

    Science.gov (United States)

    Synesiou, Elena; Fairbanks, Lynnette D; Simmonds, H Anne; Slominska, Ewa M; Smolenski, Ryszard T; Carrey, Elizabeth A

    2011-06-01

    We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells.

  6. 4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate (4PyTP, a Novel NAD+ Metabolite Accumulating in Erythrocytes of Uremic Children: A Biomarker for a Toxic NAD+ Analogue in Other Tissues?

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Carrey

    2011-06-01

    Full Text Available We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP, which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD+ metabolites (nicotinamide, N1-methylnicotinamide and 4Py-riboside and the major nicotinamide metabolite N1-methyl-2-pyridone-5-carboxamide (2PY, with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD+ from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD+ analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD+ analogue that inhibits IMP dehydrogenase in other cells.

  7. Restoration of uridine 5′-triphosphate-suppressed delayed rectifying K+ currents by an NO activator KMUP-1 involves RhoA/Rho kinase signaling in pulmonary artery smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Zen-Kong Dai

    2016-12-01

    Full Text Available We have demonstrated that KMUP-1 (7-[2-[4-(2-chlorobenzenepiperazinyl]ethyl]-1,3-dimethylxanthine blunts monocrotaline-induced pulmonary arterial hypertension by altering Ca2+ sensitivity, K+-channel function, endothelial nitric oxide synthase activity, and RhoA/Rho kinase (ROCK expression. This study further investigated whether KMUP-1 impedes uridine 5′-triphosphate (UTP-inhibited delayed rectifying K+ (KDR current in rat pulmonary arteries involved the RhoA/ROCK signaling. Pulmonary artery smooth muscle cells (PASMCs were enzymatically dissociated from rat pulmonary arteries. KMUP-1 (30μM attenuated UTP (30μM-mediated membrane depolarization and abolished UTP-enhanced cytosolic Ca2+ concentration. Whole-cell patch-clamp electrophysiology was used to monitor KDR currents. A voltage-dependent KDR current was isolated and shown to consist of a 4-aminopyridine (5mM-sensitive component and an insensitive component. The 4-aminopyridine sensitive KDR current was suppressed by UTP (30μM. The ROCK inhibitor Y27632 (30μM abolished the ability of UTP to inhibit the KDR current. Like Y27632, KMUP-1 (30μM similarly abolished UTP-inhibited KDR currents. Superfused protein kinase A and protein kinase G inhibitors (KT5720, 300nM and KT5823, 300nM did not affect UTP-inhibited KDR currents, but the currents were restored by adding KMUP-1 (30μM to the superfusate. KMUP-1 reversal of KDR current inhibition by UTP predominantly involves the ROCK inhibition. The results indicate that the RhoA/ROCK signaling pathway plays a key role in eliciting PASMCs depolarization caused by UTP, which would result in pulmonary artery constriction. KMUP-1 blocks UTP-mediated PASMCs depolarization, suggesting that it would prevent abnormal pulmonary vasoconstriction.

  8. 4-Alkyloxyimino derivatives of uridine-5'-triphosphate: distal modification of potent agonists as a strategy for molecular probes of P2Y2, P2Y4, and P2Y6 receptors.

    Science.gov (United States)

    Jayasekara, P Suresh; Barrett, Matthew O; Ball, Christopher B; Brown, Kyle A; Hammes, Eva; Balasubramanian, Ramachandran; Harden, T Kendall; Jacobson, Kenneth A

    2014-05-08

    Extended N(4)-(3-arylpropyl)oxy derivatives of uridine-5'-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N(4)-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N(4)-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N(4)-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.

  9. Variations in Cellular Responses of Mouse T Cells to Adenosine-5′-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage

    Directory of Open Access Journals (Sweden)

    Hanaa Safya

    2018-02-01

    Full Text Available A previous report has shown that regulatory T cells (Treg were markedly more sensitive to adenosine-5′-triphosphate (ATP than conventional T cells (Tconv. Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS. In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the

  10. Variations in Cellular Responses of Mouse T Cells to Adenosine-5′-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage

    Science.gov (United States)

    Safya, Hanaa; Mellouk, Amine; Legrand, Julie; Le Gall, Sylvain M.; Benbijja, Mohcine; Kanellopoulos-Langevin, Colette; Kanellopoulos, Jean M.; Bobé, Pierre

    2018-01-01

    A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5′-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different

  11. Variations in Cellular Responses of Mouse T Cells to Adenosine-5'-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage.

    Science.gov (United States)

    Safya, Hanaa; Mellouk, Amine; Legrand, Julie; Le Gall, Sylvain M; Benbijja, Mohcine; Kanellopoulos-Langevin, Colette; Kanellopoulos, Jean M; Bobé, Pierre

    2018-01-01

    A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5'-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RB low Tconvs, but not CD45RB high Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RB low Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RB high CD44 low to effector/memory CD45RB low CD44 high stage. Maximum levels of upregulation are reached on recently activated CD69 + naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69 + CD45RB high CD44 low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RB high CD44 low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RB low Tconvs, CD45RB low Foxp3 + Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary

  12. Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses

    International Nuclear Information System (INIS)

    Neumueller, M.K.; Karlstroem, A.R.K.; Kaellander, C.F.G.; Gronowitz, J.S.

    1990-01-01

    A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin

  13. In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver Efecto in vivo de adenosina 5´-trifosfato sobre el hígado preneoplásico de la rata

    Directory of Open Access Journals (Sweden)

    Ana V. Frontini

    2011-04-01

    Full Text Available The utilization of adenosine 5´-triphosphate (ATP infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.La utilización de adenosina 5´-trifosfato (ATP para inhibir el crecimiento de algunos tumores en humanos y en animales se basa en la actividad anticancerígena observada en experimentos in vitro e in vivo. El uso del ATP en la práctica clínica es discutido debido a resultados contradictorios. Por otra parte, no existen antecedentes del uso de ATP en el tratamiento de hepatocarcinomas. El objetivo del presente estudio fue determinar si el ATP previene la oncogénesis in vivo en un modelo de

  14. Alteration of radiation response in vitro by simultaneous modulation of the de novo and salvage pathways to deoxynucleoside triphosphate pool by (E)-2'-deoxy-(fluoromethylene)cytidine and thymidine analogs

    International Nuclear Information System (INIS)

    Coucke, Philippe A.; Li Yexiong; Cottin, Eliane; Ozsahin, Mahmut; Paschoud, Nicolas; Mirimanoff, Rene-Oliver

    1997-01-01

    Aim: To test different thymidine analogs (zidovudine = AZT, stavudine D4T, and 5-iodo-2-deoxyuridine = IdUrd), active at the level of the salvage pathway, as modifiers of the radiosensitization observed with (E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC), an inhibitor of ribonucleotide reductase (RR), a key enzyme in the de novo pathway of deoxynucleoside triphosphate synthesis. The thymidine anologs are interacting with thymidine kinase (TK), the rate-limiting enzyme of the salvage pathway. Materials and methods: A human colon cancer cell line Widr has been exposed for 48 h prior to irradiation to 30 nM of (E)-2'-Deoxy-(fluoromethylene)cytidine (FMdC). Stavudine (D4T) and zidovudine (AZT) were added at a concentration of 25 μM after subcultivation at low density and immediately before irradiation. Iododeoxyuridine (IdUrd) at a concentration of 1 and 2 μM was tested either 48 h before irradiation or applied in the same experimental conditions as D4T or AZT. All the concentrations used were in the physiological range. Prolonged exposure to IdUrd prior to irradiation was tested because of its known radiosensitizing effect. The experimental endpoint was clonogenic survival as measured by a colony forming assay. Results: The different drugs used alone or in combination did not modify significantly the PE of unirradiated WiDr-cells. As previously shown 30 nM FMdC for 48 h, resulted in a small but significant change in the radiation response. The enhancement ratio (SF%-control divided by SF%-drug) at a 2 Gy dose level was 1.20 ± 0.03 for cells exposed to FMdC. AZT and D4T added after subcultivation before irradiation yielded an ER of 1.12 ± 0.05 and 1.13 ± 0.09 respectively. The combination of FMdC and D4T resulted in an ER of 1.39 ± 0.04, whereas AZT combined to FMdC yielded 1.64 ± 0.21. For IdUrd alone, an ER was reached of 1.17 ± 0.05, 1.22 ± 0.08 at 1 and 2 μM respectively if applied immediately after subcultivation, and 1.17 ± 0.06 at 1 μM applied for 48

  15. Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes.

    Science.gov (United States)

    Odagaki, Yuji; Kinoshita, Masakazu; Toyoshima, Ryoichi

    2014-03-05

    Functional coupling between serotonin2A (5-HT2A) receptors and Gαq proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5׳-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-Gαq antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-Gαq antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [35S]GTPγS binding to Gαq in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of Gαq proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (±)-baclofen. When 5-HT2A receptors and M1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of Gαq proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via Gαq proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M1 receptors.

  16. UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli

    International Nuclear Information System (INIS)

    Das, S.K.; Loeb, L.A.

    1984-01-01

    UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP. These selective increases in dATP and dTTP pools are seen in wild-type E. coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose. The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed. (orig.)

  17. Adenosine triphosphate turnover in humans. Decreased degradation during relative hyperphosphatemia

    International Nuclear Information System (INIS)

    Johnson, M.A.; Tekkanat, K.; Schmaltz, S.P.; Fox, I.H.

    1989-01-01

    The regulation of ATP metabolism by inorganic phosphate (Pi) was examined in five normal volunteers through measurements of ATP degradation during relative Pi depletion and repletion states. Relative Pi depletion was achieved through dietary restriction and phosphate binders, whereas a Pi-repleted state was produced by oral Pi supplementation. ATP was radioactively labeled by the infusion of [8( 14 C)]adenine. Fructose infusion was used to produce rapid ATP degradation during Pi depletion and repletion states. Baseline measurements indicated a significant decrease of Pi levels during phosphate depletion and no change in serum or urinary purines. Serum values of Pi declined 20 to 26% within 15 min after fructose infusion in all states. Urine measurements of ATP degradation products showed an eightfold increase within 15 min after fructose infusion in both Pi-depleted and -supplemented states. Urinary radioactive ATP degradation products were fourfold higher and urinary purine specific activity was more than threefold higher during Pi depletion as compared with Pi repletion. Our data indicate that there is decreased ATP degradation to purine end products during a relative phosphate repletion state as compared to a relative phosphate depletion state. These data show that ATP metabolism can be altered through manipulation of the relative Pi state in humans

  18. Microcystin-LR acute exposure does not alter in vitro and in vivo ATP, ADP and AMP hydrolysis in adult zebrafish (Danio rerio brain membranes

    Directory of Open Access Journals (Sweden)

    Luiza Wilges Kist

    2012-10-01

    Full Text Available Microcystins (MCs are toxins produced by cyanobacteria during the blooms that could accumulate in aquatic animals and be relocated to higher trophic levels. Adenosine triphosphate (ATP acts as an excitatory neurotransmitter and/or a neuromodulator in the extracellular space playing important roles in physiological and pathological conditions. The aim of this study was, therefore, to evaluate the acute effects of different concentrations of MC-LR on nucleoside triphosphate diphosphohydrolases and 5’-nucleotidade in adult zebrafish (Danio rerio brain membranes. The results have shown no significant changes in ATP, adenosine diphosphate (ADP and adenosine monophosphate (AMP hydrolysis in zebrafish brain membranes. MC-LR in vitro also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidase activity in the conditions tested. However, additional studies including chronic exposure should be performed in order to achieve a better understanding about MC-LR toxicity mechanisms in the central nervous system.

  19. Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2 and immunolocalisation of this protein in the Schistosoma mansoni egg

    Directory of Open Access Journals (Sweden)

    Rita Gabriela Pedrosa Ribeiro Mendes

    2011-11-01

    Full Text Available A peptide (SmB2LJ; r175-194 that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

  20. The ATP/ADP substrate specificity switch between Toxoplasma gondii NTPDase1 and NTPDase3 is caused by an altered mode of binding of the substrate base.

    Science.gov (United States)

    Krug, Ulrike; Totzauer, Robert; Zebisch, Matthias; Sträter, Norbert

    2013-11-25

    Two nucleoside triphosphate diphosphohydrolase isoforms (NTPDase1 and NTPDase3) are responsible for the hydrolysis of nucleotides by the intracellular protozoan Toxoplasma gondii. They constitute about 3 % of the total parasite protein. Despite sharing 97 % sequence identity they exhibit opposite ATP versus ADP substrate discrimination ratios. Here we show by mutagenesis that the residues G492/G493 in NTPDase3 and R492/E493 in NTPDase1 are predominantly responsible for the differences in substrate specificity. Crystal structures of NTPDase1 in complexation with analogues of ATP and ADP reveal that the inverted substrate specificity of NTPDase1 relative to NTPDase3 is achieved by switching from the canonical substrate binding mode to a very different alternative one. Instead of being stacked on top of a helix of the C-terminal domain the nucleotide base is positioned in the interdomain space between the side chains of R108 and R492, recruited from both domains. Furthermore, we show that the NTPDase1 substrate specificity is mainly dependent on the presence of the side chain of E493, which causes repositioning of the ribose component of the nucleotide. All in all, binding by the flexible side chains in the alternative binding mode in NTPDase1 allows for equally good positioning of ATP and ADP with increased activity toward ADP relative to what is seen in the case of NTPDase3. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Caffeic acid treatment alters the extracellular adenine nucleotide hydrolysis in platelets and lymphocytes of adult rats.

    Science.gov (United States)

    Anwar, Javed; Spanevello, Roselia Maria; Pimentel, Victor Camera; Gutierres, Jessié; Thomé, Gustavo; Cardoso, Andreia; Zanini, Daniela; Martins, Caroline; Palma, Heloisa Einloft; Bagatini, Margarete Dulce; Baldissarelli, Jucimara; Schmatz, Roberta; Leal, Cláudio Alberto Martins; da Costa, Pauline; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

    2013-06-01

    This study evaluated the effects of caffeic acid on ectonucleotidase activities such as NTPDase (nucleoside triphosphate diphosphohydrolase), Ecto-NPP (nucleotide pyrophosphatase/phosphodiesterase), 5'-nucleotidase and adenosine deaminase (ADA) in platelets and lymphocytes of rats, as well as in the profile of platelet aggregation. Animals were divided into five groups: I (control); II (oil); III (caffeic acid 10 mg/kg); IV (caffeic acid 50 mg/kg); and V (caffeic acid 100 mg/kg). Animals were treated with caffeic acid diluted in oil for 30 days. In platelets, caffeic acid decreased the ATP hydrolysis and increased ADP hydrolysis in groups III, IV and V when compared to control (P<0.05). The 5'-nucleotidase activity was decreased, while E-NPP and ADA activities were increased in platelets of rats of groups III, IV and V (P<0.05). Caffeic acid reduced significantly the platelet aggregation in the animals of groups III, IV and V in relation to group I (P<0.05). In lymphocytes, the NTPDase and ADA activities were increased in all groups treated with caffeic acid when compared to control (P<0.05). These findings demonstrated that the enzymes were altered in tissues by caffeic acid and this compound decreased the platelet aggregation suggesting that caffeic acid should be considered a potentially therapeutic agent in disorders related to the purinergic system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Bodipy-Labeled Nucleoside Triphosphates for Polymerase Synthesis of Fluorescent DNA

    Czech Academy of Sciences Publication Activity Database

    Dziuba, Dmytro; Pohl, Radek; Hocek, Michal

    2014-01-01

    Roč. 25, č. 11 (2014), s. 1984 -1995 ISSN 1043-1802 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : enzyme amplification reaction * thymidine nucleotide analog * living cells Subject RIV: CC - Organic Chemistry Impact factor: 4.513, year: 2014

  3. Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers.

    Science.gov (United States)

    1986-01-01

    cells is diminished (406,432), a finding consistent with the decreased deformability and increased fragility of erythrocytes in Duchenne patients (170...Berne, T.W. Rall and R. Rubio, eds. The Hague: Martinus Nijhoff. pp. 179-201, 1983. - 347. Plagemann, P.G.W., Wohlhueter, R.M. and Erbe , J. Nucleoside...in Duchenne and myotonic muscular dystrophy. Nature 268:55-56, 1977. I5 . e. -. 𔃺 6 246 407. Sparks, Jr., H.V., and Belloni, F.L. The peripheral

  4. Adenosine triphosphate analogs can efficiently inhibit the Zika virus RNA-dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Hercík, Kamil; Kozák, Jaroslav; Šála, Michal; Dejmek, Milan; Hřebabecký, Hubert; Zborníková, Eva; Smola, Miroslav; Růžek, Daniel; Nencka, Radim; Bouřa, Evžen

    2017-01-01

    Roč. 137, Jan (2017), s. 131-133 ISSN 0166-3542 R&D Projects: GA ČR GA15-09310S Institutional support: RVO:61388963 ; RVO:60077344 Keywords : hepatitis C virus * borne encephalitis virus * crystal structure Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.271, year: 2016

  5. Beneficial effects of adenosine triphosphate-sensitive K+ channel opener on liver ischemia/reperfusion injury.

    Science.gov (United States)

    Nogueira, Mateus Antunes; Coelho, Ana Maria Mendonça; Sampietre, Sandra Nassa; Patzina, Rosely Antunes; Pinheiro da Silva, Fabiano; D'Albuquerque, Luiz Augusto Carneiro; Machado, Marcel Cerqueira Cesar

    2014-11-07

    To investigate the effect of diazoxide administration on liver ischemia/reperfusion injury. Wistar male rats underwent partial liver ischemia performed by clamping the pedicle from the medium and left anterior lateral segments for 1 h under mechanical ventilation. They were divided into 3 groups: Control Group, rats submitted to liver manipulation, Saline Group, rats received saline, and Diazoxide Group, rats received intravenous injection diazoxide (3.5 mg/kg) 15 min before liver reperfusion. 4 h and 24 h after reperfusion, blood was collected for determination of aspartate transaminase (AST), alanine transaminase (ALT), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), nitrite/nitrate, creatinine and tumor growth factor-β1 (TGF-β1). Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidase (MPO) were also determined. Four hours after reperfusion the diazoxide group presented with significant reduction of AST (2009 ± 257 U/L vs 3523 ± 424 U/L, P = 0.005); ALT (1794 ± 295 U/L vs 3316 ± 413 U/L, P = 0.005); TNF-α (17 ± 9 pg/mL vs 152 ± 43 pg/mL, P = 0.013; IL-6 (62 ± 18 pg/mL vs 281 ± 92 pg/mL); IL-10 (40 ± 9 pg/mL vs 78 ± 10 pg/mL P = 0.03), and nitrite/nitrate (3.8 ± 0.9 μmol/L vs 10.2 ± 2.4 μmol/L, P = 0.025) when compared to the saline group. A significant reduction in liver mitochondrial dysfunction was observed in the diazoxide group compared to the saline group (P < 0.05). No differences in liver MDA content, serum creatinine, pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion the diazoxide group showed a reduction of AST (495 ± 78 U/L vs 978 ± 192 U/L, P = 0.032); ALT (335 ± 59 U/L vs 742 ± 182 U/L, P = 0.048), and TGF-β1 (11 ± 1 ng/mL vs 17 ± 0.5 ng/mL, P = 0.004) serum levels when compared to the saline group. The control group did not present alterations when compared to the diazoxide and saline groups. Diazoxide maintains liver mitochondrial function, increases liver tolerance to ischemia/reperfusion injury, and reduces the systemic inflammatory response. These effects require further evaluation for using in a clinical setting.

  6. Evolutionary, kinetic and thermodynamic aspects on the bioenergetics of inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP)

    International Nuclear Information System (INIS)

    Baltscheffsky, H.; Baltscheffsky, M.

    1995-01-01

    Energy barriers for energy carriers are of fundamental significance for the successful operation of the bioenergetic reactions in living cells. PPi and ATP are outstanding ''energy-rich'' examples of molecular ''energy currencies'' in biological systems, with kinetic barriers preventing excessively fast thermodynamically feasible hydrolysis from occurring. The barriers may be considered to facilitate the energy coupling roles of these phosphate compounds, which are to secure growth and maintain numerous other energy requiring functions. The enzymes involved in overcoming the energies of activation of the bioenergetic reactions have evolved to be very well tuned for their roles. Three aspects will be discussed in some detail. The first is the fact that ATP at neutral pH is considerably more energy-rich than PPi, which thus has been called a ''poor man's ATP''. This is exemplified by the kinetic and thermodynamic differences observed between the requirements for the photosynthetic formation of PPi and ATP in certain photobacterial chromatophores by varying levels of energy supply. At lower pH, PPi and ATP are equally energy-rich, which may be of significance for acidophiles. The second concerns the possible evolutionary significance of the finding that, in the dark, a pH gradient suffices to drive extensive PPi synthesis, whereas ATP synthesis requires both a pH gradient and a membrane potential (Strid et al, Biochim. Biophys. Acta 892 (1987) 236-244). Thirdly, PPi as the most plausible predecessor to ATP in the origin and early evolution of life, will be discussed. (author). Abstract only

  7. Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms.

    Science.gov (United States)

    Mondal, Subhasish; Roy, Jay Jyoti; Bera, Tanmoy

    2014-03-01

    Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.

  8. Effect of different superovulation stimulation protocols on adenosine triphosphate concentration in rabbit oocytes.

    Science.gov (United States)

    Cortell, Carmela; Salvetti, Pascal; Joly, Thierry; Viudes-de-Castro, Maria Pilar

    2015-08-01

    Ovarian stimulation protocols are used usually to increase the number of oocytes collected. The determination of how oocyte quality may be affected by these superovulation procedures, therefore, would be very useful. There is a high correlation between oocyte ATP concentration and developmental competence of the resulting embryo. The aim of this study was to evaluate the effect of follicle stimulating hormone (FSH) origin and administration protocols on oocyte ATP content. Rabbit does were distributed randomly into four groups: (i) a control group; (ii) the rhFSH3 group: females were injected, every 24 h over 3 days, with 0.6 μl of rhFSH diluted in polyvinylpyrrolidone (PVP); (iii) the pFSH3 group: females were injected every 24 h over 3 days with 11.4 μg of pFSH diluted in PVP; and (iv) the pFSH5 group: females were injected twice a day for 5 days with 11.4 μg of pFSH diluted in saline serum. Secondly, the effect of pFSH5 protocol on developmental potential was evaluated. Developmental competence of oocytes from the control and pFSH5 groups was examined. Differences in superovulation treatments were found for ATP levels. In the pFSH5 group, the ATP level was significantly lower than that of the other groups (5.63 ± 0.14 for pFSH group versus 6.42 ± 0.13 and 6.19 ± 0.15 for rhFSH3 and pFSH3, respectively; P ATP production, it is possible that, after this superovulation treatment, oocyte metabolism would be affected.

  9. Influence of intracellular adenosine-triphosphate concentration of yeast cells on survival following X-irradiation

    International Nuclear Information System (INIS)

    Reinhard, R.D.; Pohlit, W.

    1975-01-01

    The effect of D-glucose, 2-deoxy-D-glucose and starvation in buffer on the ATP-concentration of yeast cells has been studied. In both the wild-type and a respiratory-deficient mutant strain 2-deoxy-D-glucose decreases the value for ATP, while it is enhanced by glucose only in the mutant strain. Populations with different ATP-concentrations have been irradiated. The results suggest that ATP may be an essential factor in the system that determines the length of the shoulder of the dose effect curves. (orig.) [de

  10. Synthesis and enzymatic incorporation of α-L-threofuranosyl adenine triphosphate (tATP).

    Science.gov (United States)

    Zhang, Su; Chaput, John C

    2013-03-01

    Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Therminator DNA polymerase to copy DNA templates into TNA. Here, we expand the substrate repertoire of Therminator DNA polymerase to include threofuranosyl adenine 3'-triphsophate (tATP). We chemically synthesized tATP by two different methods from the 2'-O-acetyl derivative. Enzyme-mediated polymerization reveals that tATP functions as an efficient substrate for Therminator DNA polymerase, indicating that tATP can replace the diaminopurine analogue (tDTP) in TNA transcription reactions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Comparison of the novel vasodilator uridine triphosphate and adenosine for the measurement of fractional flow reserve

    DEFF Research Database (Denmark)

    Sivertsen, Jacob; Jensen, Jan Skov; Galatius, Søren

    2014-01-01

    and IC infusion (using a microcatheter in the coronary ostium). Standard IV adenosine infusion (140 μg/kg/min) was compared to 8 equimolar incremental doses of IC UTP and IC adenosine (20, 40, 60, 80, 160, 240, 320 and 640 μg/min) in a randomized order. Across all doses, ΔFFR[IC UTP - IC adenosine...

  12. Mechanisms by Which Human DNA Primase Chooses To Polymerize a Nucleoside Triphosphate

    Czech Academy of Sciences Publication Activity Database

    Urban, M.; Joubert, Nicolas; Purse, B. W.; Hocek, Michal; Kuchta, R. D.

    2010-01-01

    Roč. 49, č. 4 (2010), s. 727-735 ISSN 0006-2960 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550902 Grant - others:NIH(US) GM54194 Institutional research plan: CEZ:AV0Z40550506 Keywords : C-nucleosides * DNA polymerase * primase * mechanism Subject RIV: CC - Organic Chemistry Impact factor: 3.226, year: 2010

  13. Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates

    DEFF Research Database (Denmark)

    Abraham, E H; Shrivastav, B; Salikhova, A Y

    2001-01-01

    P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated...... provides a framework for understanding the role of erythrocytes in drug resistance. The erythrocyte consists of a membrane surrounding a millimolar pool of ATP. Mammalian RBCs have no nucleus or DNA drug/toxin targets. From the perspective of drug/ATP complex formation, the RBC serves as an important...

  14. Increased deoxythymidine triphosphate levels is a feature of relative cognitive decline

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Frederiksen, Jane H; Olsen, Maria Nathalie Angleys

    2015-01-01

    Mitochondrial bioenergetics, mitochondrial reactive oxygen species (ROS) and cellular levels of nucleotides have been hypothesized as early indicators of Alzheimer's disease (AD). Utilizing relative decline of cognitive ability as a predictor of AD risk, we evaluated the correlation between chang...

  15. Adenosine triphosphate is a critical determinant for VEGFR signal during hypoxia.

    Science.gov (United States)

    Al Mamun, Abdullah; Hayashi, Hisaki; Sakima, Miho; Sato, Motohiko

    2016-12-01

    Hypoxia induces angiogenesis through the VEGF signaling pathway; however, signal propagation of VEGF in hypoxia is not fully understood. In this study, we examined alterations in VEGF signaling during hypoxia conditions and its determinant in endothelial cells. To analyze VEGF signaling during hypoxia, human umbilical vein endothelial cells (HUVECs) were exposed to 3 h of hypoxia (1% O 2 ) followed by 3 h of reoxygenation or 12 h of hypoxia. Hypoxia induced expression of VEGF mRNA, but it was not associated with an increase in tube formation by HUVECs. During 3 h of hypoxia, VEGF-induced phosphorylation of VEGF receptor-2 (VEGFR-2) and downstream molecules were significantly inhibited without a change in VEGFR-2 expression, but it was completely restored after reoxygenation. VEGF-mediated VEGFR-2 phosphorylation is associated with a reduction in cellular ATP in hypoxia conditions (65.93 ± 8.32% of normoxia, means ± SE, P determinant of VEGF signaling in hypoxia and suggest that the activation process of VEGFR-2 was modified by sustained hypoxia. These observations contribute to our understanding of signal alterations in VEGF in endothelial cells during hypoxia. Copyright © 2016 the American Physiological Society.

  16. A modified method for synthesis of [γ-32P] labelled adenosine triphosphate

    International Nuclear Information System (INIS)

    Rahman, W.Y.; Rohadi Awaludin; Endang Sarmini; Herlina; Triyanto; Rien Ritawidya; Abdul Mutalib; Santi Nurbaiti

    2015-01-01

    Production of [γ- 32 P]-ATP using three glycolysis enzymatic reaction i.e. glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglyceric phosphokinase and lactate dehydrogenase has been conducted. dl-glyceraldehyde 3-phosphate, Adenosine Diphosphate and H 3 32 PO 4 was used as precursors for this reaction. Purification of [γ- 32 P]-ATP was performed by using DEAE-Sephadex column chromatography. The result suggested that this simple method could be used for producing [γ- 32 P]-ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia. (author)

  17. Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Koka, P.

    1984-01-01

    A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

  18. Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major

    DEFF Research Database (Denmark)

    Camacho, A; Arrebola, R; Pena Diaz, Javier

    1997-01-01

    A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli. The cDNA contained an open reading frame that encoded a protein of 269 amino...... complemented dUTPase deficiency in Escherichia coli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability...

  19. Coxsackievirus cloverleaf RNA containing a 5' triphosphate triggers an antiviral response via RIG-I activation

    NARCIS (Netherlands)

    Feng, Qian; Langereis, Martijn A; Olagnier, David; Chiang, Cindy; van de Winkel, Roel; van Essen, Peter; Zoll, Jan; Hiscott, John; van Kuppeveld, Frank J M

    2014-01-01

    Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by

  20. Glyphosate-based herbicide affects biochemical parameters in Rhamdia quelen (Quoy & Gaimard, 1824 and Leporinus obtusidens (Valenciennes, 1837

    Directory of Open Access Journals (Sweden)

    Vania Lucia Loro

    Full Text Available Rhamdia quelen (silver catfish and Leporinus obtusidens (piava were exposed to a commercial formulation Roundup(r, a glyphosate-based herbicide at concentrations of 0.2 or 0.4 mg/L for 96 h. The effects of the herbicide were analyzed on the alanine aminotransferase (ALT and aspartate aminotransferase (AST activities and glucose in plasma, glucose and protein in the mucus layer, nucleotide hydrolysis in the brain, and protein carbonyl in the liver. The parameters were chosen, owing to a lack of information concerning integrated analysis, considering oxidative damage parameters, liver damage, and effects on the mucus layer composition and triphosphate diphosphohydrolase (NTPDase activities. Plasmatic glucose levels were reduced in both species, whereas the transaminase activities (ALT and AST increased after exposure to the herbicide. Herbicide exposure increased protein and glucose levels in the mucus layer in both species. There was a reduction in both NTPDase and ecto-5'-nucleotidase activity in the brain of piava, and increased enzyme activity in silver catfish at both concentrations tested. The species showed an increase in protein carbonyl in the liver after exposure to both concentrations of the glyphosate. Our results demonstrated that exposure to Roundup(r caused liver damage, as evidenced by increased plasma transaminases and liver protein carbonyl in both of the fish species studied. The mucus composition changed and hypoglycemia was detected after Roundup(r exposure in both species. Brain nucleotide hydrolysis showed a different response for each fish species studied. These parameters indicated some important and potential indicators of glyphosate contamination in aquatic ecosystems.

  1. Piracetam prevents scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities.

    Science.gov (United States)

    Marisco, Patricia C; Carvalho, Fabiano B; Rosa, Michelle M; Girardi, Bruna A; Gutierres, Jessié M; Jaques, Jeandre A S; Salla, Ana P S; Pimentel, Víctor C; Schetinger, Maria Rosa C; Leal, Daniela B R; Mello, Carlos F; Rubin, Maribel A

    2013-08-01

    Piracetam improves cognitive function in animals and in human beings, but its mechanism of action is still not completely known. In the present study, we investigated whether enzymes involved in extracellular adenine nucleotide metabolism, adenosine triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase and adenosine deaminase (ADA) are affected by piracetam in the hippocampus and cerebral cortex of animals subjected to scopolamine-induced memory impairment. Piracetam (0.02 μmol/5 μL, intracerebroventricular, 60 min pre-training) prevented memory impairment induced by scopolamine (1 mg/kg, intraperitoneal, immediately post-training) in the inhibitory avoidance learning and in the object recognition task. Scopolamine reduced the activity of NTPDase in hippocampus (53 % for ATP and 53 % for ADP hydrolysis) and cerebral cortex (28 % for ATP hydrolysis). Scopolamine also decreased the activity of 5'-nucleotidase (43 %) and ADA (91 %) in hippocampus. The same effect was observed in the cerebral cortex for 5'-nucleotidase (38 %) and ADA (68 %) activities. Piracetam fully prevented scopolamine-induced memory impairment and decrease of NTPDase, 5'-nucleotidase and adenosine deaminase activities in synaptosomes from cerebral cortex and hippocampus. In vitro experiments show that piracetam and scopolamine did not alter enzymatic activity in cerebral cortex synaptosomes. Moreover, piracetam prevented scopolamine-induced increase of TBARS levels in hippocampus and cerebral cortex. These results suggest that piracetam-induced improvement of memory is associated with protection against oxidative stress and maintenance of NTPDase, 5'-nucleotidase and ADA activities, and suggest the purinergic system as a putative target of piracetam.

  2. Expression of adenosine triphosphate-sensitive potassium channels in rats with cirrhosis: correlationship with sympathetic activity and renal function

    Directory of Open Access Journals (Sweden)

    Julio Cesar Martins Monte

    2006-12-01

    Full Text Available Objective: The aim of this study was to perform a direct analysis ofKATP mRNA expression by RT-PCR in kidney and isolated aorta fromrats with cirrhosis (induced by carbon tetrachloride and controls.The present study also analyses the relation between induced cirrhosisand urinary excretion of sodium and sympathetic activity in cirrhoticrats. Methods: Rats were placed in metabolic cages and allowedfree access to food and water. Cirrhosis was induced by repeateddoses of carbon tetrachloride by gastric gavage. After some weeks,the kidney and aorta were dissected and utilized for RNA extraction.Blood and urine were analyzed for electrolytes. Renal function wasestimated by creatinine clearance and sodium urinary excretion.Serum catecholamines were measured by HPLC analysis. Results:First, RT-PCR analysis showed that KATP mRNA is expressed in liverwith cirrhosis and intense fibrosis, but not with moderate fibrosis.Second, RT-PCR analysis revealed that KATP mRNA was detectedonly in aorta dissected from rats with cirrhosis. Finally, an enhancedreabsorption of sodium without renal failure suggests a potentialmediator would increase the activity of the sympathetic system.Conclusion: These results suggest that KATP mRNA is expressed incirrhotic rats with sympathetic activation and renal dysfunction. Thischannel might be involved in another route where the vascular tonecan be modulated in cirrhosis.

  3. Rosuvastatin lowers coenzyme Q10 levels, but not mitochondrial adenosine triphosphate synthesis, in children with familial hypercholesterolemia

    NARCIS (Netherlands)

    Avis, Hans J.; Hargreaves, Ian P.; Ruiter, Jos P. N.; Land, John M.; Wanders, Ronald J.; Wijburg, Frits A.

    2011-01-01

    To investigate whether statin therapy affects coenzyme Q10 (CoQ10) status in children with heterozygous familial hypercholesterolemia (FH). Samples were obtained at baseline (treatment naïve) and after dose titration with rosuvastatin, aiming for a low-density lipoprotein cholesterol level of 110

  4. Synthesis of nucleoside mono- and triphosphates bearing oligopyridine ligands, their incorporation into DNA and complexation with transition metals

    Czech Academy of Sciences Publication Activity Database

    Kalachová, Lubica; Pohl, Radek; Hocek, Michal

    2012-01-01

    Roč. 10, č. 1 (2012), s. 49-55 ISSN 1477-0520 R&D Projects: GA ČR GA203/09/0317; GA MŠk LC512 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotides * terpyridine * DNA Subject RIV: CC - Organic Chemistry Impact factor: 3.568, year: 2012

  5. A simplified method for rapid quantification of intracellular nucleoside triphosphates by one-dimensional thin-layer chromatography

    DEFF Research Database (Denmark)

    Jendresen, Christian Bille; Kilstrup, Mogens; Martinussen, Jan

    2011-01-01

    -pyrophosphate (PRPP), and inorganic pyrophosphate (PPi) in cell extracts. The method uses one-dimensional thin-layer chromatography (TLC) and radiolabeled biological samples. Nucleotides are resolved at the level of ionic charge in an optimized acidic ammonium formate and chloride solvent, permitting...... quantification of NTPs. The method is significantly simpler and faster than both current two-dimensional methods and high-performance liquid chromatography (HPLC)-based procedures, allowing a higher throughput while common sources of inaccuracies and technical problems are avoided. For determination of PPi...

  6. Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates. Synthesis, polymerase incorporation to DNA and electrochemical study

    Czech Academy of Sciences Publication Activity Database

    Macíčková-Cahová, Hana; Pohl, Radek; Horáková Brázdilová, Petra; Havran, Luděk; Špaček, Jan; Fojta, Miroslav; Hocek, Michal

    2011-01-01

    Roč. 17, č. 21 (2011), s. 5833-5841 ISSN 0947-6539 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA ČR GA203/09/0317; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA polymerases * electrochemistry * nucleosides * nucleotides * organosulfur compounds Subject RIV: CC - Organic Chemistry Impact factor: 5.925, year: 2011

  7. Electrochemical behavior of anthraquinone- and nitrophenyl-labeled deoxynucleoside triphosphates: a contribution to development of multipotential redox labeling of DNA

    Czech Academy of Sciences Publication Activity Database

    Vidláková, Pavlína; Pivoňková, Hana; Fojta, Miroslav; Havran, Luděk

    2015-01-01

    Roč. 146, č. 5 (2015), s. 839-847 ISSN 0026-9247 R&D Projects: GA ČR(CZ) GBP206/12/G151; GA ČR(CZ) GAP206/12/2378 Institutional support: RVO:68081707 Keywords : SOLID AMALGAM ELECTRODE * OSMIUM-TETROXIDE COMPLEXES * AQUEOUS-METHANOL MEDIUM Subject RIV: BO - Biophysics Impact factor: 1.131, year: 2015

  8. Enzymatic Recognition of 2′‐Modified Ribonucleoside 5′‐Triphosphates: Towards the Evolution of Versatile Aptamers

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Rothnagel, Joseph A.; Veedu, Rakesh N.

    2012-01-01

    The quest for effective, selective and nontoxic nucleic‐acid‐based drugs has led to designing modifications of naturally occurring nucleosides. A number of modified nucleic acids have been made in the past decades in the hope that they would prove useful in target‐validation studies and therapeutic...... and investigated as substrates for various enzymes. Herein, we review studies on the enzyme‐recognition of various 2′‐sugar‐modified NTPs that were carried out with a view to their effective utilization in SELEX processes to generate versatile aptamers....

  9. Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5′-triphosphate Synthesis in Chloroplasts 1

    Science.gov (United States)

    Queirolo, Carlos B.; Andreo, Carlos S.; Vallejos, Rubén H.; Niemeyer, Hermann M.; Corcuera, Luis J.

    1981-01-01

    Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-β-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF1) and of purified CF1. On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation. PMID:16662030

  10. Induced hypothermia is protective in a rat model of pneumococcal pneumonia associated with increased adenosine triphosphate availability and turnover

    NARCIS (Netherlands)

    Beurskens, Charlotte J. P.; Aslami, Hamid; Kuipers, Maria T.; Horn, Janneke; Vroom, Margreeth B.; van Kuilenburg, André B. P.; Roelofs, Joris J. T. H.; Schultz, Marcus J.; Juffermans, Nicole P.

    2012-01-01

    Objective: To determine the effect of induced hypothermia on bacterial growth, lung injury, and mitochondrial function in a rat model of pneumococcal pneumosepsis. Design: Animal study. Setting: University research laboratory. Subjects: Male Sprague-Dawley rats. Interventions: Subjects were

  11. Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Hocek, Michal; Fojta, Miroslav

    2008-01-01

    Roč. 6, č. 13 (2008), s. 2233-2241 ISSN 1477-0520 R&D Projects: GA ČR GA203/05/0043; GA ČR GA203/07/1195; GA MŠk LC512; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507 Keywords : DNA * nucleotides * cross-coupling reactions Subject RIV: CC - Organic Chemistry Impact factor: 3.550, year: 2008

  12. Stereospecific recognition sites for [3H]inositol(1,4,5)-triphosphate in particulate preparations of rat cerebellum

    International Nuclear Information System (INIS)

    Willcocks, A.L.; Cooke, A.M.; Potter, B.V.; Nahorski, S.R.

    1987-01-01

    A very high density of stereospecific binding sites for inositol-(1,4,5)P3 have been identified in rat cerebellar membranes using [ 3 H]inositol-(1,4,5)P3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (KD 23 nM). Incubations were carried out at 4 degrees and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [ 3 H]-(1,4,5)P3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P3 was the only other phosphate to show relatively high affinity (KD 1500 nM). HPLC-pure Ins(1,3,4)P3 and Ins(1,3,4,5)P4 were substantially weaker and Ins(1,4)P2, Ins-2-P1, Ins-1-P1, Ins(1,2)-cyclic P1 and inositol were totally inactive at concentrations less than 50 microM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P3 can initiate the release of bound Ca 2+

  13. Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities †

    OpenAIRE

    Karl, D. M.; Craven, D. B.

    1980-01-01

    Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, w...

  14. New luminescence-based approach to measurement of luciferase gene expression reporter activity and adenosine triphosphate-based determination of cell viability

    Czech Academy of Sciences Publication Activity Database

    Konopka, Roman; Hýžďalová, Martina; Kubala, Lukáš; Pacherník, J.

    2010-01-01

    Roč. 56, č. 2 (2010), s. 66-71 ISSN 0015-5500 R&D Projects: GA ČR(CZ) GA524/06/1197 Grant - others:GA ČR(CZ) GA301/08/0717 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : cell viability * luciferase * reporter gene assay Subject RIV: BO - Biophysics Impact factor: 0.729, year: 2010

  15. Pacific ciguatoxin-1b effect over Na+ and K+ currents, inositol 1,4,5-triphosphate content and intracellular Ca2+ signals in cultured rat myotubes

    Science.gov (United States)

    Hidalgo, Jorge; Liberona, José Luis; Molgó, Jordi; Jaimovich, Enrique

    2002-01-01

    The action of the main ciguatoxin involved in ciguatera fish poisoning in the Pacific region (P-CTX-1b) was studied in myotubes originated from rat skeletal muscle cells kept in primary culture. The effect of P-CTX-1b on sodium currents at short times of exposure (up to 1 min) showed a moderate increase in peak Na+ current. During prolonged exposures, P-CTX-1b decreased the peak Na+ current. This action was always accompanied by an increase of leakage currents, tail currents and outward Na+ currents, resulting in an intracellular Na+ accumulation. This effect is blocked by prior exposure to tetrodotoxin (TTX) and becomes evident only after washout of TTX. Low to moderate concentrations of P-CTX-1b (2–5 nM) partially blocked potassium currents in a manner that was dependent on the membrane potential. P-CTX-1b (2–12 nM) caused a small membrane depolarization (3–5 mV) and an increase in the frequency of spontaneous action potential discharges that reached in general low frequencies (0.1–0.5 Hz). P-CTX-1b (10 nM) caused a transient increase of intracellular inositol 1,4,5-trisphosphate (IP3) mass levels, which was blocked by TTX. In the presence of P-CTX-1b (10 nM) and in the absence of external Ca2+, the intracellular Ca2+ levels show a transient increase in the cytoplasm as well as in the nuclei. The time course of this effect may reflect the action of IP3 over internal stores activated by P-CTX-1b-induced membrane depolarization. PMID:12429578

  16. Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774

    Energy Technology Data Exchange (ETDEWEB)

    Gavel, Olga Yu.; Kladova, Anna V.; Bursakov, Sergey A.; Dias, João M.; Texeira, Susana; Shnyrov, Valery L.; Moura, José J. G.; Moura, Isabel; Romão, Maria J.; Trincão, José, E-mail: trincao@dq.fct.unl.pt [REQUIMTE, Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

    2008-07-01

    Native zinc-containing ATP sulfurylase from D. desulfuricans ATCC 27774 was purified to homogeneity and crystallized. Diffraction data were collected to 2.5 Å resolution. Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 Å resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

  17. Aqueous Heck Cross-Coupling Preparation of Acrylate-Modified Nucleotides and Nucleoside Triphosphates for Polymerase Synthesis of Acrylate-Labeled DNA

    Czech Academy of Sciences Publication Activity Database

    Daďová, Jitka; Vidláková, Pavlína; Pohl, Radek; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2013-01-01

    Roč. 78, č. 19 (2013), s. 9627-9637 ISSN 0022-3263 R&D Projects: GA AV ČR(CZ) IAA400040901 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : nucleic-acids * enzymatic incorporation * protein interactions * functionalized DNA Subject RIV: CC - Organic Chemistry Impact factor: 4.638, year: 2013

  18. Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release.

    Science.gov (United States)

    Cook, Zara C; Gray, Michael A; Cann, Martin J

    2012-07-27

    Elevated CO(2) is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO(2) blunts G protein-activated cAMP signaling. The effect of CO(2) is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO(2) on cAMP levels required the activity of the IP(3) receptor. Consistent with these findings, CO(2) caused an increase in steady state cytoplasmic Ca(2+) concentrations not observed in the absence of the IP(3) receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na(+)/H(+) antiporter (NHE3) to demonstrate a functional relevance for CO(2)-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO(2) abrogated the inhibitory effect of cAMP on NHE3 function via an IP(3) receptor-dependent mechanism.

  19. Elevated Carbon Dioxide Blunts Mammalian cAMP Signaling Dependent on Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Release*

    Science.gov (United States)

    Cook, Zara C.; Gray, Michael A.; Cann, Martin J.

    2012-01-01

    Elevated CO2 is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO2 blunts G protein-activated cAMP signaling. The effect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO2 on cAMP levels required the activity of the IP3 receptor. Consistent with these findings, CO2 caused an increase in steady state cytoplasmic Ca2+ concentrations not observed in the absence of the IP3 receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na+/H+ antiporter (NHE3) to demonstrate a functional relevance for CO2-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO2 abrogated the inhibitory effect of cAMP on NHE3 function via an IP3 receptor-dependent mechanism. PMID:22654111

  20. Kinetics of UV-induced changes in deoxynucleoside triphosphate pools in Chinese hamster ovary cells and their effect on measurements of DNA synthesis.

    Science.gov (United States)

    Newman, C N; Miller, J H

    1983-11-15

    Measurements of dNTP pools following exposure of Chinese hamster ovary cells to ultraviolet radiation reveals a rapid accumulation of cellular dTTP and a rapid loss of cellular dCTP. Exposure to 3-, 10- or 20 Jm-2 results in a 3-, 4- or 5.4-fold increase in cellular dTTP, respectively, within the first 10 min after exposure. dTTP levels then decrease noticeably, approaching the control value 3 to 5 hr later. In contrast, dCTP levels decrease rapidly within 10 min after exposure, ultimately to 1/10 that observed in the unirradiated control population. Recovery to normal dCTP levels is slow, taking in excess of 12 hr. No change in dATP is observed for 1-2 hr; subsequently, a moderate decrease in dATP levels occurs which is then followed by recovery, beginning 8 hr after irradiation. These results contrast with changes in dNTP pools observed in Chinese hamster V-79 cells exposed to mutagens. Measurements of rates of DNA synthesis by pulse-labeling cells with [3H]thymidine are also apparently affected by UV-induced transient deviations in the endogenous radiospecific activity of the labeled precursor.

  1. Phosphorus-31 magnetic relaxation of adenosine 5‧-monophosphate, adenosine 5‧-diphosphate and adenosine 5‧-triphosphate in solution

    Science.gov (United States)

    Gaspar, R., Jr.; Brey, W. S., Jr.; Qiu, A.; Andrew, E. R.

    1989-04-01

    Measurements have been made of the longitudinal phosphorus-31 magnetic relaxation of the individual phosphorus resonances of AMP, ADP and ATP at 121.5 MHz from 278 to 333 K and at 40.5 MHz at room temperature in H 2O solutions of the chemicals. The phosphorus-31 spin-lattice relaxation times of all compounds are dominated by dipolar interactions and influenced by chemical shift anisotropy interactions to a different extent at the two frequencies. Phosphate group motion superimposed on the tumbling of the molecules is the main source of phosphorus-31 spin-lattice relaxation. Activation energies characterizing the combined motion range between 17.1 and 20.0 kJ/mol.

  2. An NMR study of 1H, 31P, and 23Na relaxation and molecular dynamics in the polycrystalline sodium salts of adenosine Di- and triphosphate

    Science.gov (United States)

    Reynhardt, E. C.; Jurga, K.; Andrew, E. R.

    Proton spin-lattice relaxation times in the laboratory frame, T1(H),have been measured as a function of frequency and temperature (333K> T > 80 K). The spin-lattice relaxation times in the rotating frame, T1 ϱ(H), have been measured at two different rotating fields while M2(H), the proton second moment, has been extracted from the shape of the FID. In addition, T 1( 31P) and T 1( 23Na) have been measured as functions of temperature at 81 and 50 MHz, respectively. The results demonstrate clearly that the water content of the compounds influences the results to a large extent. It seems that water molecules at some of the lattice sites can be removed from the structure by evacuation, while others are more tightly bound to the ADP and ATP molecules. The more loosely bound water molecules are very mobile and dominate the relaxation results in the high-temperature region via the spin-rotation and dipolar mechanisms. The more tightly bound water molecules rotate about their twofold axes and this motion, characterized by a distribution of correlation times, results in a T1(H) minimum in the low-temperature region. The results have been interpreted in terms of a Fuoss-Kirkwood distribution function. The 23Na spin-lattice relaxation rates are dominated by the quadrupolar interactions, which provide a dominating relaxation mechanism for the proton spins in the rotating frame. In the case of Na 2ATP, T1(P) is independent of the degree of hydration of the sample, but the NaADP T1(H), values are influenced strongly by a change in the water content. An X-ray determination of the lengths of the a axes of the unit cells has provided supporting evidence for the interpretation of the NMR results.

  3. Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 mono- and bis-(M)ANT-substituted nucleoside 5'-triphosphates.

    Science.gov (United States)

    Taha, Hesham; Dove, Stefan; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Seifert, Roland

    2012-01-01

    Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins, i.e. lethal factor, protective antigen and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). Conventional antibiotic treatment is ineffective against either toxaemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Our previous studies showed that EF is differentially inhibited by various purine and pyrimidine nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl (ANT) groups at the 2'(3')-O-ribosyl position, with the unique preference for the base cytosine (Taha et al., Mol Pharmacol 75:693 (2009)). MANT-CTP was the most potent EF inhibitor (K (i), 100 nM) among 16 compounds studied. Here, we examined the interaction of EF with a series of 18 2',3'-O-mono- and bis-(M)ANT-substituted nucleotides, recently shown to be very potent inhibitors of the AC toxin from Bordetella pertussis, CyaA (Geduhn et al., J Pharmacol Exp Ther 336:104 (2011)). We analysed purified EF and EF mutants in radiometric AC assays and in fluorescence spectroscopy studies and conducted molecular modelling studies. Bis-MANT nucleotides inhibited EF competitively. Propyl-ANT-ATP was the most potent EF inhibitor (K (i), 80 nM). In contrast to the observations made for CyaA, introduction of a second (M)ANT-group decreased rather than increased inhibitor potency at EF. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to bis-MANT-ATP, but FRET to bis-MANT-CTP was only small. Mutations N583Q, K353A and K353R differentially altered the inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP. The nucleotide binding site of EF accommodates bulky bis-(M)ANT-substituted purine and pyrimidine nucleotides, but the fit is suboptimal compared to CyaA. These data provide a basis for future studies aiming at the development of potent EF inhibitors with high selectivity relative to mammalian ACs.

  4. Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes.

    Science.gov (United States)

    Gong, Xue; Li, Jinfu; Zhou, Wenjiao; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-05-30

    Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Enzyme-linked electrochemical detection of DNA fragments amplified by PCR in the presence of a biotinylated deoxynucleoside triphosphate using disposable pencil graphite electrodes

    Czech Academy of Sciences Publication Activity Database

    Haroniková, Lucia; Špaček, Jan; Plucnara, Medard; Horáková, Petra; Pivoňková, Hana; Havran, Luděk; Erdem, A.; Fojta, Miroslav

    2015-01-01

    Roč. 146, č. 5 (2015), s. 849-855 ISSN 0026-9247 R&D Projects: GA ČR(CZ) GAP206/11/1638; GA ČR(CZ) GPP206/11/P739 Institutional support: RVO:68081707 Keywords : POLYMERASE-CHAIN-REACTION * V-LEIDEN MUTATION * SENSITIVE DETECTION Subject RIV: BO - Biophysics Impact factor: 1.131, year: 2015

  6. Molecular mechanisms behind the accumulation of adenosine triphosphate (ATP) and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection

    Science.gov (United States)

    Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells,...

  7. Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

    Science.gov (United States)

    Walpole, Thomas B; Palmer, David N; Jiang, Huibing; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2015-04-01

    The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate

    International Nuclear Information System (INIS)

    Xu, K.Y.; Kyte, J.

    1989-01-01

    An immunoadsorbent specific for the carboxy-terminal sequence -GAPER, which comprises residues 502-506 of the alpha-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase], was used to isolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H]acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys501, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E1 conformation or when the structure of the enzyme was changed from the E1 conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H]acetyl suggest that Lys501 from the alpha polypeptide is directly involved in binding ATP within the active site of (Na+ + K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodies raised against the sequence-GAPER and denatured or enzymically active (Na+ + K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the alpha-polypeptide, respectively

  9. 2'-(R)-Fluorinated mC, hmC, fC and caC triphosphates are substrates for DNA polymerases and TET-enzymes.

    Science.gov (United States)

    Schröder, A S; Parsa, E; Iwan, K; Traube, F R; Wallner, M; Serdjukow, S; Carell, T

    2016-12-13

    A deeper investigation of the chemistry that occurs on the newly discovered epigenetic DNA bases 5-hydroxymethyl-(hmdC), 5-formyl-(fdC), and 5-carboxy-deoxycytidine (cadC) requires chemical tool compounds, which are able to dissect the different potential reaction pathways in cells. Here we report that the 2'-(R)-fluorinated derivatives F-hmdC, F-fdC, and F-cadC, which are resistant to removal by base excision repair, are good substrates for DNA polymerases and TET enzymes. This result shows that the fluorinated compounds are ideal tool substances to investigate potential C-C-bond cleaving reactions in the context of active demethylation.

  10. Variants of the inosine triphosphate pyrophosphatase gene are associated with reduced relapse risk following treatment for HCV genotype 2/3

    DEFF Research Database (Denmark)

    Rembeck, Karolina; Waldenström, Jesper; Hellstrand, Kristoffer

    2014-01-01

    studies using higher, weight-based ribavirin dosing report that patients carrying polymorphisms encoding reduced predicted ITPase activity show decreased risk of ribavirin-induced anemia but increased risk of thrombocytopenia, with no impact on elimination of virus. Three hundred fifty-four treatment...... duration arms, HCV genotype, fibrosis stage and IL28B genotype, and was not secondary to improved adherence to therapy or less pronounced anemia. Gene variants predicting reduced predicted ITPase activity also were associated with decreased risk of anemia (P....007), and lower ribavirin concentrations (P=0.02). Conclusion: These findings demonstrate a novel ribavirin-like association between polymorphisms at ITPA and treatment efficacy in chronic hepatitis C mediated by reduced relapse risk. We hypothesize that patients (63%) being homozygous for both major alleles...

  11. Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

    International Nuclear Information System (INIS)

    Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

    1988-01-01

    The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [ 32 P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-α/sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-α/sub i/ or anti-α 0 antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed

  12. Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl tranferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

    Czech Academy of Sciences Publication Activity Database

    Horáková Brázdilová, Petra; Macíčková-Cahová, Hana; Pivoňková, Hana; Špaček, Jan; Havran, Luděk; Hocek, Michal; Fojta, Miroslav

    2011-01-01

    Roč. 9, č. 5 (2011), s. 1366-1371 ISSN 1477-0520 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk(CZ) LC512; GA AV ČR(CZ) IAA400040901 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : DNA tail-labelling * protein-DNA binding * DNA hybridization Subject RIV: BO - Biophysics Impact factor: 3.696, year: 2011

  13. Dideoxynucleoside triphosphate-sensitive DNA polymerase from rice is involved in base excision repair and immunologically similar to mammalian DNA pol beta.

    Science.gov (United States)

    Sarkar, Sailendra Nath; Bakshi, Sankar; Mokkapati, Sanath K; Roy, Sujit; Sengupta, Dibyendu N

    2004-07-16

    A single polypeptide with ddNTP-sensitive DNA polymerase activity was purified to near homogeneity from the shoot tips of rice seedlings and analysis of the preparations by SDS-PAGE followed by silver staining showed a polypeptide of 67 kDa size. The DNA polymerase activity was found to be inhibitory by ddNTP in both in vitro DNA polymerase activity assay and activity gel analysis. Aphidicolin, an inhibitor of other types of DNA polymerases, had no effect on plant enzyme. The 67 kDa rice DNA polymerase was found to be recognized by the polyclonal antibody (purified IgG) made against rat DNA polymerase beta (pol beta) both in solution and also on Western blot. The recognition was found to be very specific as the activity of Klenow enzyme was unaffected by the antibody. The ability of rice nuclear extract to correct G:U mismatch of oligo-duplex was observed when oligo-duplex with 32P-labeled lower strand containing U (at 22nd position) was used as substrate. Differential appearance of bands at 21-mer, 22-mer, and 51-mer position in presence of dCTP was visible only with G:U mismatch oligo-duplex, but not with G:C oligo-duplex. While ddCTP or polyclonal antibody against rat-DNA pol beta inhibits base excision repair (BER), aphidicolin had no effect. These results for the first time clearly demonstrate the ability of rice nuclear extract to run BER and the involvement of ddNTP-sensitive pol beta type DNA polymerase. Immunological similarity of the ddNTP-sensitive DNA polymerase beta of rice and rat and its involvement in BER revealed the conservation of structure and function of ddNTP-sensitive DNA pol beta in plant and animal.

  14. Ca2+-mediated generation of inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K+, glucose, and carbamylcholine

    International Nuclear Information System (INIS)

    Biden, T.J.; Peter-Riesch, B.; Schlegel, W.; Wollheim, C.B.

    1987-01-01

    The role of Ca2+ in the generation of inositol phosphates was investigated using rat pancreatic islets after steady state labeling with myo-[2- 3 H]inositol. Depolarizing K+ concentrations (24 mM) evoked early (2 s) increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) as measured by high performance anion-exchange chromatography. The increase in Ins-1,4,5-P3 was transient and was followed by a more pronounced rise in Ins-1,3,4-P3. These effects were dependent on the presence of extracellular Ca2+ but were not secondary to release of either neurotransmitters or metabolites of arachidonic acid. K+ also promoted the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) and of the other phosphoinositides. Glucose (16.7 mM) was less marked in its effects but still promoted rapid increases in Ins-1,3,4,5-P4 (2 s) and Ins-1,4,5-P3 (10 s) and a slower rise in Ins-1,3,4-P3 (30 s). The levels of all three metabolites rose steadily over 10 min stimulation. These responses to glucose could be largely, although not entirely, inhibited by depletion of extracellular Ca2+ or by Ca2+ channel blockade with verapamil (20 microM). Carbamylcholine (0.5 mM) was the most potent stimulus used evoking early rises in Ins-1,4,5-P3 and Ins-1,3,4,5-P4 (2 s) followed by Ins-1,3,4-P3 (10 s), effects which were only partially dependent on extracellular Ca2+. The results suggest that a Ca2+-mediated PtdIns-4,5-P2 hydrolysis accounts for most of the Ins-1,4,5-P3 generated in response to glucose but not carbamylcholine

  15. Active-site modification of mammalian DNA polymerase β with pyridoxal 5'-phosphate: Mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

    International Nuclear Information System (INIS)

    Basu, A.; Kedar, P.; Wilson, S.H.; Modak, M.J.

    1989-01-01

    Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase β. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [ 3 H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate

  16. UP-REGULATION OF ANTITHROMBOTIC ECTONUCLEOTIDASES BY ASPIRIN IN HUMAN ENDOTHELIAL-CELLS IN-VITRO

    NARCIS (Netherlands)

    CHEUNG, PK; VISSER, J; BAKKER, WW

    1994-01-01

    Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP

  17. Profil Kinetik dan Efektivitas Enrofloksasin yang Dikombinasikan dengan BioATP dalam Mengatasi Coxiella burnetii (KINETIC PROFILE AND EFFECTIVITY OF ENROFLOXACINE WITH BIO ADENOSIN TRIPHOSPHATE SUPPLEMENTATION AGAINST COXIELLA BURNETII

    Directory of Open Access Journals (Sweden)

    Andriyanto .

    2013-11-01

    Full Text Available Coxiella burnetii belongs to rikettsia group living obligate intracellularly and as the agent of zoonosisQ fever. Enrofloxacine is an antibiotic in quinolon group used to treat infection of C. burnetii in chicken,goat, calve, pig, dog, cat,  and horse. From ruminant practical experience, enrofloxacine if combined withBioATP  can enhance the enrofloxacine activity. Research for the effecivity of enrofloxacine and BioATP totreat C. burnetii has never been carried out. The research was conducted to explore effect of enrofloxacinewith supplementation BioATP against C. burnetii. Enrofloxacine pharmacokinetic study was carried outby using simental beef as an experimental animals. The effectivity of BioATP supplementation onenrofloxacine activity to treat C. burnetii was tested by using Vero cell tissue culture. The results showedthat combination of enrofloxacine and BioATP increased kinetic profile of enrofloxacine in term of onset,duration, pharmacology intensity, and bioavailaibility. Enrofloxacine had activity to treat C. burnetii withvalue of minimal inhibitory concentration (MIC at 1-2 ppm and value of minimal bactericidal concentrationat 4 ppm. Supplementation of BioATP improved the effectivity of enrofloxacine in treating C. burnetii.

  18. Genetic Susceptibility of Lung Cancer Associated With Common Variants in the 3 ' Untranslated Regions of the Adenosine Triphosphate-Binding Cassette B1 (ABCB1) and ABCC1 Candidate Transporter Genes for Carcinogen Export

    NARCIS (Netherlands)

    Wang, Haijian; Jin, Guangfu; Wang, Haifeng; Liu, Gaifen; Qian, Ji; Jin, Li; Wei, Qingyi; Shen, Hongbing; Huang, Wei; Lu, Daru

    2009-01-01

    BACKGROUND: Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NKK) is a well defined carcinogen that can induce lung cancer. Genetic polymorphisms in its disposition pathways could modify the risk of developing lung cancer. The authors of this report previously catalogued

  19. 6-Aryl-4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (benzo-fused 7-deaza-dATP analogues): Synthesis, fluorescent properties, enzymatic incorporation into DNA and DNA-protein binding study

    Czech Academy of Sciences Publication Activity Database

    Bosáková, A.; Perlíková, Pavla; Tichý, Michal; Pohl, Radek; Hocek, Michal

    2016-01-01

    Roč. 24, č. 19 (2016), s. 4528-4535 ISSN 0968-0896 R&D Projects: GA ČR(CZ) GA16-00178S Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidoindoles * deazapurines * fluorescence * DNA polymerase Subject RIV: CC - Organic Chemistry Impact factor: 2.930, year: 2016

  20. The Dynamics of the Microbial Population as Measured by the Quantification of adenosine 5'-triphosphate (ATP) at Three Sampling Locations Within the North Inlet Estuary, Georgetown, SC: 1981-1985.

    Data.gov (United States)

    Baruch Institute for Marine and Coastal Sciences, Univ of South Carolina — Water samples were collected daily at approximately 10:00 AM, from a depth of 50 cm at three stations, and transported immediately to the laboratory. The three...

  1. Use of actin-bound adenosine 5'-diphosphate as a method to determine the specific 32P-radioactivity of the gamma-phosphoryl group of adenosine 5'-triphosphate in a highly compartmentalized cell, the platelet

    International Nuclear Information System (INIS)

    Verhoeven, A.J.; Cook, C.A.; Holmsen, H.

    1988-01-01

    Determination of the specific 32 P-radioactivity of cytoplasmic ATP in 32 P-Pi-labeled platelets is complicated by the presence of a large pool of metabolically inactive, granule-stored nucleotides. Moreover, our data show that the specific 32 P-radioactivity of cytoplasmic ATP is severely underestimated when determined in platelets after the complete secretion of granule-stored nucleotides, possibly due to isotopic dilution with granule-stored phosphate. As F-actin-bound ADP is ethanol-insoluble, this pool can be readily separated from the other nucleotide pools in platelets. Here we show that the specific 32 P-radioactivity of F-actin-bound ADP accurately reflects that of the gamma-phosphoryl group of cytoplasmic ATP. During uptake of 32 P-Pi by human platelets the specific 32 P-radioactivity of F-actin-bound ADP equals that of the monoester phosphates of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, which are in metabolic equilibrium with cytoplasmic ATP. Therefore, this method enables the determination of the specific 32 P-radioactivity of the gamma-phosphoryl group of cytoplasmic ATP in platelets even under short-term labeling conditions

  2. Anatomy of the primase-alpha DNA polymerase reaction accomplished by nucleoprotein complexes harboring an exrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenil deoxyguanosine-5-triphosphate

    Czech Academy of Sciences Publication Activity Database

    Říman, Josef

    2001-01-01

    Roč. 45, č. 1 (2001), s. 109-124 ISSN 0001-723X Institutional research plan: CEZ:AV0Z5052915 Keywords : primase * alpha DNA-polymerase * enzyme complex Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.644, year: 2001

  3. Cross-coupling reactions of unprotected halopurine bases, nucleosides, nucleotides and nucleoside triphosphates with 4-boronophenylalanine in water. Synthesis of (purin-8-yl)- and (purin-6-yl)phenylalanines

    Czech Academy of Sciences Publication Activity Database

    Čapek, Petr; Pohl, Radek; Hocek, Michal

    2006-01-01

    Roč. 4, č. 11 (2006), s. 2278-2284 ISSN 1477-0520 R&D Projects: GA AV ČR(CZ) 1QS400550501; GA MŠk(CZ) 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : amino acids * purines * nucleosides * cross-coupling reactions Subject RIV: CC - Organic Chemistry Impact factor: 2.874, year: 2006

  4. Chemoattractant and guanosine 5'-[γ-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Vries, Martinus J. de; Penning, Louis C.; Roovers, Edwin; Kaay, Jeroen van der; Erneux, Christophe; Lookeren Campagne, Michiel M. van

    1989-01-01

    The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol

  5. Development of a human-specific B. thetaiotaomicron IMS/ATP assay for measuring viable human contamination in surface waters in Baja California, Mexico

    Science.gov (United States)

    Immunomagnetic separation/adenosine triphosphate (IMS/ATP) assays utilize paramagnetic beads and target-specific antibodies to isolate target organisms. Following isolation, adenosine tri-phosphate (ATP) is extracted from the target population and quantified. An inversely-couple...

  6. Muscle 3243A-->G mutation load and capacity of the mitochondrial energy-generating system

    NARCIS (Netherlands)

    Janssen, A.J.M.; Schuelke, M.; Smeitink, J.A.M.; Trijbels, F.J.M.; Sengers, R.C.A.; Lucke, B.; Wintjes, L.T.M.; Morava, E.; van Engelen, B.G.M.; Smits, B.W.; Hol, F.A.; Siers, M.H.; Ter Laak, H.; van der Knaap, M.S.; van Spronsen, F.J.; Rodenburg, R.J.T.; van den Heuvel, L.P.

    2008-01-01

    Objective: The mitochondrial energy-generating system (MEGS) encompasses the mitochondrial enzymatic reactions from oxidation of pyruvate to the export of adenosine triphosphate. It is investigated in intact muscle mitochondria by measuring the pyruvate oxidation and adenosine triphosphate

  7. Hasan, Prof. Gaiti

    Indian Academy of Sciences (India)

    Cerebellar Ataxia, Neurodegeneration, Molecular Genetics, Drosophila, Calcium Signalling in Neurons, Inositol Triphosphate Receptor Address: Professor, National Centre for Biological Sciences, GKVK Campus, Bengaluru 560 065, Karnataka

  8. Cyclopentenyl cytosine induces apoptosis and increases cytarabine-induced apoptosis in a T-lymphoblastic leukemic cell-line

    NARCIS (Netherlands)

    Verschuur, A. C.; Brinkman, J.; van Gennip, A. H.; Leen, R.; Vet, R. J.; Evers, L. M.; Voûte, P. A.; van Kuilenburg, A. B.

    2001-01-01

    Cyclopentenyl cytosine (CPEC) is a nucleoside-analogue that decreases the concentrations of cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in leukemic cells by inhibiting the enzyme CTP synthetase, resulting in a decreased synthesis of RNA and DNA. Low concentrations of dCTP

  9. Childhood lymphoblastic leukemia adverse drug reactions: study of risk factors and therapy prognosis by optical methods

    Science.gov (United States)

    Zyubin, A.; Lavrova, A.; Babak, S.; Malaschenko, V.; Borisova, A.; Opryshko, N.

    2016-10-01

    The treatment of acute lymphoblastic leukemia (ALL) can result in the side-effects such as kidney affection, hepatic failure and tissue hypoxia. We study dynamics of special biochemical marker of these pathologies - adenosine triphosphate, that is well-known substance of energy metabolism. We use methods of confocal microscopy for determining the cellular and mitochondrial concentration of adenosine triphosphate (ATP). Quantitative values of adenosine triphosphate were calculated for each patient and correlation with degree of side-effects had been done. The application of confocal microscopy for studying of side-effects and therapy of lymphoblastic leukemia is discussed.

  10. ORF Alignment: NC_004337 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_004337 gi|24113675 >1nbuA 1 118 5 120 2e-19 ... ref|NP_708185.1| D-erythro-7,8-dihydrone...opterin triphosphate epimerase [Shigella ... flexneri 2a str. 301] gb|AAN43892.1| ... D-erythro-7,8-dihydrone....1| ... D-erythro-7,8-dihydroneopterin triphosphate epimerase ... [...Shigella flexneri 2a str. 2457T] ref|NP_754732.1| ... D-erythro-7,8-dihydroneopterin triphosphate epi...merase ... [Escherichia coli CFT073] gb|AAP17710.1| ... D-erythro-7,8-dihydroneopterin triphos

  11. Dna Sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  12. Studies towards the synthesis of ATP analogs as potential glutamine synthetase inhibitors

    CSIR Research Space (South Africa)

    Salisu, S

    2011-05-01

    Full Text Available In research directed at the development of adenine triphosphate (ATP) analogs as potential glutamine synthetase (GS) inhibitors, adenine and allopurinol derivatives have been synthesized either as novel ATP analogs or as scaffolds...

  13. Hypoxia-induced tumor cell resistance is overcome by synergistic GAPDH-siRNA and chemotherapy co-delivered by long-circulating and cationic-interior liposomes

    NARCIS (Netherlands)

    Guan, J.; Sun, J.; Sun, F.|info:eu-repo/dai/nl/370549775; Lou, B.|info:eu-repo/dai/nl/37052537X; Zhang, D.; Mashayekhi, V.|info:eu-repo/dai/nl/413278360; Sadeghi, N.; Storm, G.|info:eu-repo/dai/nl/073356328; Mastrobattista, E.|info:eu-repo/dai/nl/228061105; He, Z.

    2017-01-01

    Chemotherapeutic drug resistance of tumor cells under hypoxic conditions is caused by the inhibition of apoptosis by autophagy and drug efflux via adenosine triphosphate (ATP)-dependent transporter activation, among other factors. Here, we demonstrate that disrupting glyceraldehyde-3-phosphate

  14. Reference: 735 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available aura et al. 2008 Apr. Plant Physiol. 146(4):1721-37. Oxidative respiration produces adenosine triphosphate t...nses to mitochondrial electron transport. 4 1721-37 18305213 2008 Apr Plant physio

  15. 1164-IJBCS-Article-Mrs Gboko Ouattara A

    African Journals Online (AJOL)

    T60

    Naseri and Heidari,. 2007) which in turn, increases the intracellular calcium though inositol triphosphate (IP3). (Naseri and Heidari, 2007) and also by facilitating the inflow of extracellular calcium though the receptor-operated calcium channel.

  16. Creatine kinase activity is associated with blood pressure

    NARCIS (Netherlands)

    Brewster, Lizzy M.; Mairuhu, Gideon; Bindraban, Navin R.; Koopmans, Richard P.; Clark, Joseph F.; van Montfrans, Gert A.

    2006-01-01

    BACKGROUND: We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular

  17. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  18. Effects of ketamine and its isomers on ischemic preconditioning in the isolated rat heart

    NARCIS (Netherlands)

    Molojavyi, A.; Preckel, B.; Comfère, T.; Müllenheim, J.; Thämer, V.; Schlack, W.

    2001-01-01

    BACKGROUND: Ischemic preconditioning protects the heart against subsequent ischemia. Opening of the adenosine triphosphate-sensitive potassium (KATP) channel is a key mechanism of preconditioning. Ketamine blocks KATP channels of isolated cardiomyocytes. The authors investigated the effects of

  19. Hasan, Prof. Gaiti

    Indian Academy of Sciences (India)

    Specialization: Transcriptomics, Spino-Cerebellar Ataxia, Neurodegeneration, Molecular Genetics, Drosophila, Calcium Signalling in Neurons, Inositol Triphosphate Receptor Address: Professor, National Centre for Biological Sciences, GKVK Campus, Bengaluru 560 065, Karnataka Contact: Office: (080) 2366 6140

  20. Red blood cell phosphate concentration and osmotic resistance during dietary phosphate depletion in dairy cows

    NARCIS (Netherlands)

    Grünberg, W; Mol, J A; Teske, E

    BACKGROUND: Hypophosphatemia in early lactating dairy cows has been implicated as primary cause for postparturient hemoglobinuria in cattle. Decreased availability of phosphorus has been proposed to reduce adenosine triphosphate synthesis of erythrocytes and thereby reduce osmotic resistance of

  1. Redox-sensitive alteration of replisome architecture safeguards genome integrity

    DEFF Research Database (Denmark)

    Somyajit, Kumar; Gupta, Rajat; Sedlackova, Hana

    2017-01-01

    DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX...

  2. An Investigation of the Potential of 31-Phosphorus Nuclear Magnetic Resonance Spectroscopy to Predict Radiation Sensitivity

    Science.gov (United States)

    1988-01-01

    adenine triphosphate (NTP). The PME resonance originates primarily from phosphorylethanolamine (PE) and phosphorylcholine (PC). The phosphate diester (PDE...1985. 17. Gyulai, L., Bolinger, L., Leigh, J. S., Jr., Barlow, C., and Chance, B. Phosphorylethanolamine -the major constituent of the

  3. Rapid detection of E. Coli O157:H7 by IFAST and ATP bioluminescence assay for water analysis

    CSIR Research Space (South Africa)

    Ngamsom, B

    2016-10-01

    Full Text Available The present investigation reports isolation and detection of E. coli O157:H7 employing a simple and portable microfluidic device based on immiscible filtration assisted by surface tension (IFAST) and adenosine triphosphate (ATP) bioluminescence...

  4. Physical mechanisms of biological molecular motors

    International Nuclear Information System (INIS)

    Miller, John H. Jr.; Vajrala, Vijayanand; Infante, Hans L.; Claycomb, James R.; Palanisami, Akilan; Fang Jie; Mercier, George T.

    2009-01-01

    Biological motors generally fall into two categories: (1) those that convert chemical into mechanical energy via hydrolysis of a nucleoside triphosphate, usually adenosine triphosphate, regarded as life's chemical currency of energy and (2) membrane bound motors driven directly by an ion gradient and/or membrane potential. Here we argue that electrostatic interactions play a vital role for both types of motors and, therefore, the tools of physics can greatly contribute to understanding biological motors

  5. Stanovení adenosintrifosfátu a adenosindifosfátu v reálných vzorcích

    OpenAIRE

    Černá, Martina

    2013-01-01

    The aim of the diploma thesis was to find optimal conditions of high pressure liquid chromatography for the detection and quantification of two common nucleotides, namely adenosine diphosphate and adenosine triphosphate, as well as to perform an analysis of these in real life samples of citrus fruits and plant extracts. Further aim of the project was to determine the limits of detection and quantification of adenosine diphosphate and adenosine triphosphate under the optimized conditions and u...

  6. The diagnostic value of immunohistochemically detected methylthioadenosine phosphorylase deficiency in malignant pleural mesotheliomas

    DEFF Research Database (Denmark)

    Zimling, Zarah Glad; Jørgensen, Anne; Santoni-Rugiu, Eric

    2012-01-01

      Malignant pleural mesothelioma (MPM) often causes diagnostic difficulties for pathologists. We assessed whether loss of methylthioadenosine phosphorylase (MTAP), a key enzyme in the intracellular recycling of adenosine triphosphate (ATP) often deleted in MPM, could be detected with immunohistoc......  Malignant pleural mesothelioma (MPM) often causes diagnostic difficulties for pathologists. We assessed whether loss of methylthioadenosine phosphorylase (MTAP), a key enzyme in the intracellular recycling of adenosine triphosphate (ATP) often deleted in MPM, could be detected...

  7. Directed DNA Metallization: Towards the Construction of Rationally Designed Conductive Nanodevices

    Science.gov (United States)

    Burley, Glenn A.; Gierlich, Johannes; Hammond, David M.; Gramlich, Phillipp M. E.; Carell, Thomas

    2006-09-01

    Genes of interest can be selectively metallized via the incorporation of modified triphosphates. These triphosphates bear functions that can be further derivatised with aldehyde groups via the use of click chemistry. Treatment of the aldehyde-labeled gene mixture with the Tollens reagent, followed by a development process results in the selective metallization of the gene of interest in the presence of natural DNA strands.

  8. A parallel synthesis scheme for generating libraries of DNA polymerase substrates and inhibitors.

    Science.gov (United States)

    Strobel, Heike; Dugué, Laurence; Marlière, Philippe; Pochet, Sylvie

    2002-12-02

    We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5'-triphosphate derivative of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH(2))TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dY(NHR)TP. Vent (exo-) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dY(NHben)TP and, to a lesser extent, dY(NHphe)TP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.

  9. Effect of oocyte quality on blastocyst development after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in a sheep model.

    Science.gov (United States)

    Catalá, Maria G; Izquierdo, Dolors; Rodríguez-Prado, Maria; Hammami, Sondes; Paramio, Maria-Teresa

    2012-04-01

    To compare blastocyst production, after IVF and ICSI, from sheep oocytes of various quality. Sham-injected oocytes and parthenogenetic activated oocyte groups were considered as control. Prospective experimental study. University. Three- to 6-month-old sheep. Oocyte quality was assessed with the use of brilliant cresyl blue (BCB) stain. Adenosine triphosphate content was measured. Intracytoplasmic sperm injection and IVF were performed and blastocyst development and cell numbers were analyzed. Adenosine triphosphate content, embryo development and blastomere numbers. After IVF, BCB-stained (BCB+) oocytes developed up to the blastocyst stage at higher percentages and with more cells per embryo (24.1% vs 4.0% and 69.7 vs 43.9, respectively) than unstained (BCB-) oocytes. Using intracytoplasmic sperm injection, no differences were found in blastocyst production (14.3% vs 11.8%) and number of cells per embryo (71.1 vs 54.3) between BCB+ and BCB- oocytes. Adenosine triphosphate content was higher before in vitro maturation than after in both types of oocytes. Brilliant cresyl blue-stained oocytes had more adenosine triphosphate content than BCB- oocytes. Brilliant cresyl blue-stained oocytes show more adenosine triphosphate content than BCB- oocytes. Results from IVF were affected by the oocyte quality while ICSI did not produce differences in embryo development or blastomere numbers. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Repair replication in permeabilized Escherichia coli

    International Nuclear Information System (INIS)

    Masker, W.E.; Simon, T.J.; Hanawalt, P.C.

    1975-01-01

    We have examined the modes of DNA synthesis in Escherichia coli strains made permeable to nucleoside triphosphates by treatment with toluene. In this quasi in vitro system, polymerase-I-deficient mutants exhibit a nonconservative mode of synthesis with properties expected for the resynthesis step of excision-repair. This uv-stimulated DNA synthesis can be performed by either DNA polymerase II or III and it also requires the uvrA gene product. It requires the four deoxynucleoside triphosphates; but, in contrast to the semiconservative mode, the ATP requirement can be partially satisfied by other nucleoside triphosphates. The ATP-dependent recBC nuclease is not involved. The observed uv-stimulated mode of DNA synthesis may be part of an alternate excision-repair mechanism which supplements or complements DNA-polymerase-I-dependent repair in vivo

  11. Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille

    2008-01-01

    Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha......Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme...

  12. Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

    Science.gov (United States)

    Bitensky, M.W.; Yoshida, Tatsuro

    1997-04-29

    A method is disclosed using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4 C storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4 C for prolonged periods of time is achieved by removing oxygen from the red blood cells at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate. 4 figs.

  13. Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

    Science.gov (United States)

    Bitensky, Mark W.; Yoshida, Tatsuro

    1997-01-01

    Method using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4.degree. C. storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4.degree. C. for prolonged periods of time is achieved by removing oxygen therefrom at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate.

  14. Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies

    OpenAIRE

    Jane Dixon, C; Bowler, Wayne B; Walsh, Catherine A; Gallagher, James A

    1997-01-01

    Human osteoblasts responded to the application of extracellular nucleotides, acting at P2-receptors, with increases in cytosolic free calcium concentration ([Ca2+]i).In populations of human osteoblasts, adenosine 5′-diphosphate (ADP) evoked a rise in [Ca2+]i with less than 40% of the amplitude of that induced by adenosine 5′-triphosphate (ATP).ATP and uridine 5′-triphosphate (UTP) were applied to single human osteoblasts and induced [Ca2+]i rises of comparable amplitude in every cell tested.H...

  15. Structural basis for feedback inhibition of the deoxyribonucleoside salvage pathway: Studies of the Drosophila deoxyribonucleoside kinase

    DEFF Research Database (Denmark)

    Mikkelsen, N.E.; Johansson, K.; Karlsson, A.

    2003-01-01

    Deoxyribonucleoside kinases are feedback inhibited by the final products of the salvage pathway, the deoxyribonucleoside triphosphates. In the present study, the mechanism of feedback inhibition is presented based on the crystal structure of a complex between the fruit fly deoxyribonucleoside...... kinase and its feedback inhibitor deoxythymidine triphosphate. The inhibitor was found to be bound as a bisubstrate inhibitor with its nucleoside part in the nucleoside binding site and with its phosphate groups partially occupying the phosphate donor site. The overall structure of the enzyme...... and to the primary base, Glu52, that normally is positioned close to the 5'-OH of the substrate deoxyribose....

  16. Three-dimensional structure of the large cytoplasmic H-4-H-5 loop of Na(+)/K(+)-ATPase deduced by restraint-based comparative modeling shows only one ATP binding site

    Czech Academy of Sciences Publication Activity Database

    Ettrich, Rüdiger; Melicherčík, M.; Teisinger, Jan; Ettrichová, Olga; Krumscheid, R.; Hofbauerová, Kateřina; Kvasnička, P.; Schoner, W.; Amler, Evžen

    2001-01-01

    Roč. 7, č. 6 (2001), s. 184-192 ISSN 0948-5023 R&D Projects: GA MŠk VS961410; GA ČR GA204/98/0468; GA AV ČR IAA7011801; GA ČR GA204/98/0416 Grant - others:IWTZ(DE) TSR-088-97; Volkswagen Foundation(DE) I/74679 Institutional research plan: CEZ:AV0Z5011922 Keywords : Sodium potassium adenosine triphosphate * tertiary structure * adenosine triphosphate binding site Subject RIV: BO - Biophysics Impact factor: 1.011, year: 2001

  17. The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C.

    OpenAIRE

    Wilkinson, G. F.; Purkiss, J. R.; Boarder, M. R.

    1993-01-01

    1. We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP). 2. The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADP beta S) were potent agonists (EC50 0.41 microM and 0.85 microM respectively) while beta, gamma-methylene ATP at 300 microM was not. 3....

  18. DNA synthesis in chromatin preparations from human fibroblasts infected by cytomegalovirus

    International Nuclear Information System (INIS)

    Furukawa, T.

    1980-01-01

    Chromatin prepared from ( 14 C)-thymidine pulse labelled cytomegalovirus-infected human fibroblasts 72 hours postinfection exhibited under appropriate conditions endogenous activity of ( 3 H)-thymidine triphosphate incorporation which was relatively salt-resistant and phosphonoacetic acid-sensitive. Isopycnic centrifugation of the doubly labelled DNA in CsCl revealed that cell-free incorporation occurred into viral as well as into host cell DNA. Density labelling experiments with bromodeoxyuridine triphosphate suggested the incorporation into viral DNA to be due to replicative DNA synthesis. Chromatin from infected cells contained, in addition to cellular, viral DNA polymerase activity. (author)

  19. The Shigella flexneri OmpA amino acid residues 188EVQ190 are essential for the interaction with the virulence factor PhoN2.

    Science.gov (United States)

    Scribano, Daniela; Damico, Rosanna; Ambrosi, Cecilia; Superti, Fabiana; Marazzato, Massimiliano; Conte, Maria Pia; Longhi, Catia; Palamara, Anna Teresa; Zagaglia, Carlo; Nicoletti, Mauro

    2016-12-01

    Shigella flexneri is an intracellular pathogen that deploys an arsenal of virulence factors promoting host cell invasion, intracellular multiplication and intra- and inter-cellular dissemination. We have previously reported that the interaction between apyrase (PhoN2), a periplasmic ATP-diphosphohydrolase, and the C-terminal domain of the outer membrane (OM) protein OmpA is likely required for proper IcsA exposition at the old bacterial pole and thus for full virulence expression of Shigella flexneri (Scribano et al., 2014). OmpA, that is the major OM protein of Gram-negative bacteria, is a multifaceted protein that plays many different roles both in the OM structural integrity and in the virulence of several pathogens. Here, by using yeast two-hybrid technology and by constructing an in silico 3D model of OmpA from S. flexneri 5a strain M90T, we observed that the OmpA residues 188 EVQ 190 are likely essential for PhoN2-OmpA interaction. The 188 EVQ 190 amino acids are located within a flexible region of the OmpA protein that could represent a scaffold for protein-protein interaction.

  20. Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI and pseudoxanthoma elasticum (PXE

    Directory of Open Access Journals (Sweden)

    Alexander Apschner

    2014-07-01

    Full Text Available In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf, which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1, a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin. Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord

  1. Preparation of short cytosine-modified oligonucleotides by nicking enzyme amplification reaction

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Hocek, Michal

    2012-01-01

    Roč. 48, č. 55 (2012), s. 6921-6923 ISSN 1359-7345 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : cross-coupling reactions * nucleoside triphosphates * functionalized DNA * restriction endonucleases * polymerase incorporation Subject RIV: CC - Organic Chemistry Impact factor: 6.378, year: 2012

  2. Effect of Ethanol Root Extract of Equisetum arvense (L) on Urinary ...

    African Journals Online (AJOL)

    Also in the Equisetum arvense group, plasma adrenaline and noradrenaline levels were lower than for the control group. Furthermore, increase in the levels of urinary adenosine triphosphate was smaller in Equisetum arvense group than in control group. Conclusion: This study suggests that Equisetum arvense ethanol root ...

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions...

  4. Short-term increase of plasma free fatty acids does not interfere with intrinsic mitochondrial function in healthy young men

    NARCIS (Netherlands)

    Brands, Myrte; Hoeks, Joris; Sauerwein, Hans P.; Ackermans, Mariette T.; Ouwens, Margriet; Lammers, Nicolette M.; van der Plas, Mart N.; Schrauwen, Patrick; Groen, Albert K.; Serlie, Mireille J.

    2011-01-01

    Free fatty acid (FFA)- and obesity-induced insulin resistance has been associated with disturbed mitochondrial function. Elevated plasma FFA can impair insulin-induced increase of adenosine triphosphate synthesis and downregulate the expression of genes important in the biogenesis of mitochondria in

  5. Effect of Hoodia gordonii leaf meal supplementation at finisher stage ...

    African Journals Online (AJOL)

    UNISA

    (2004) isolated P57AS3 molecule from H. gordonii, which is a steroidal glycoside with anorectic activity in animals. Intracerebroventricular injections of the purified P57AS3 in rats increased the content of adenosine triphosphate. (ATP) by 50 to 150% in the hypothalamus neurons, thus acting as an appetite suppressor.

  6. Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Raindlová, Veronika; Hocek, Michal

    2013-01-01

    Roč. 24, č. 6 (2013), s. 1081-1093 ISSN 1043-1802 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : isothermal DNA amplification * cross-coupling reactions * nucleoside triphosphates * polymerase incorporation * functionalized DNA * nucleic-acids Subject RIV: CC - Organic Chemistry Impact factor: 4.821, year: 2013

  7. Association of P2Y(2) receptor SNPs with bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients

    DEFF Research Database (Denmark)

    Wesselius, Anke; Bours, Martijn J L; Henriksen, Zanne

    2013-01-01

    The P2Y(2) receptor is a G-protein-coupled receptor with adenosine 5'-triphosphate (and UTP) as natural ligands. It is thought to be involved in bone physiology in an anti-osteogenic manner. As several non-synonymous single nucleotide polymorphisms (SNPs) have been identified within the P2Y(2...

  8. Bioluminometric assay of ATP in mouse brain: Determinant factors ...

    Indian Academy of Sciences (India)

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the ...

  9. Development of Alginate/Chitosan Microparticles for Dust Mite Allerge

    African Journals Online (AJOL)

    The highest allergen content (0.30 ± 0.07 mg/g) was obtained with 2.5 % initial allergen loading in chitosan- triphosphate (CS-TPP) microparticles. Sustained allergen release (approx. 50 % over 24 h) was observed from alginate-coated chitosan microparticles. Allergen incorporation method and initial drug-loading could ...

  10. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.W.G.; Haastert, van P.J.M.

    2004-01-01

    The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

  11. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.; van Haastert, P.J.

    2004-01-01

    The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

  12. Magnesium - distribution and basic metabolism | Olhaberry | South ...

    African Journals Online (AJOL)

    Magnesium is extensively distributed in soil, water and plants. It is essential for ehzymatic reactions requiring adenosine triphosphate, and the recommended dietary allowance in man is 5 - 10 mg/kg/d. About 50% of magnesium in man is stored in bone, where it is regulated by parathyroid hormone'and 1,25(OH)2-D3.

  13. Magnesium - distribution and basic metabolism

    African Journals Online (AJOL)

    Magnesium is extensively distributed in soil, water and plants. It is essential for ehzymatic reactions requiring adenosine triphosphate, and the recom- mended dietary allowance in man is 5 - 10 mg/kg/d. About 50% of magnesium in man is stored ~n bone, where it is regulated by parathyroid hormone'and. 1,25(OHb-D3.

  14. Inhibition of hippocampal synaptic transmission by impairment of Ral function

    DEFF Research Database (Denmark)

    Owe-Larsson, Björn; Chaves-Olarte, Esteban; Chauhan, Ashok

    2005-01-01

    Large clostridial cytotoxins and protein overexpression were used to probe for involvement of Ras-related GTPases (guanosine triphosphate) in synaptic transmission in cultured rat hippocampal neurons. The toxins TcdA-10463 (inactivates Rho, Rac, Cdc42, Rap) and TcsL-1522 (inactivates Ral, Rac, Ra...

  15. Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides

    DEFF Research Database (Denmark)

    Højland, Torben; Veedu, Rakesh N; Vester, Birte

    2012-01-01

    We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5'-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N(m), Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands...

  16. Metabolic costs of force generation for constant-frequency and catchlike-inducing electrical stimulation in human tibialis anterior muscle

    DEFF Research Database (Denmark)

    Ratkevicius, Aivaras; Quistorff, Bjørn

    2002-01-01

    at 37.5 ms. One train was delivered to the peroneal nerve every 2.5 s for 36 times under ischemic conditions. Anaerobic adenosine triphosphate (ATP) turnover was determined using 31-phosphorus magnetic resonance spectroscopy (P-MRS) of the human tibialis anterior muscle. Compared with constant...

  17. Author Details

    African Journals Online (AJOL)

    Bukar, BB. Vol 15, No 1 (2004) - Articles Effect of DL-Carnitine on tissue content of adenosine triphosphate and creatine phospate in inschaemic myocardial tissue. Abstract PDF. ISSN: 01891731. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL ...

  18. Sequence Classification: 290568 [

    Lifescience Database Archive (English)

    Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|16130954|ref|NP_417530.1| dihydroneopte...rin aldolase, also has dihydroneopterin triphosphate 2'-epimerase activity || http://www.ncbi.nlm.nih.gov/protein/16130954 ...

  19. Sequence Classification: 525310 [

    Lifescience Database Archive (English)

    Full Text Available TMB Non-TMH Non-TMB TMB Non-TMB Non-TMB >gi|62181723|ref|YP_218140.1| dihydroneopte...rin aldolase, also has dihydroneopterin triphosphate 2'-epimerase activity || http://www.ncbi.nlm.nih.gov/protein/62181723 ...

  20. Sequence Classification: 203907 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|71891851|ref|YP_277580.1| dihydrone...opterin aldolase, also has dihydroneopterin triphosphate || http://www.ncbi.nlm.nih.gov/protein/71891851 ...

  1. Sequence Classification: 155582 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|33519541|ref|NP_878373.1| dihydrone...opterin aldolase, also has dihydroneopterin triphosphate 2'-epimerase activity || http://www.ncbi.nlm.nih.gov/protein/33519541 ...

  2. Electrokinetic enrichment and detection of neuropeptide for performance monitoring

    Science.gov (United States)

    2016-06-14

    device scaling within the electrodeless insulator dielectrophoresis geometry through the application of highly Fig. 1: Biomarker enrichment in...a competitive electrochemical detection system that is free of wash -steps and enables the real-time monitoring of adenosine triphosphate (ATP) over...methodology for quantifying the region of interest by using a statistical description of particle distribution across the device geometry to determine

  3. Electrokinetic Enrichment and Detection of Neuropeptide for Performance Monitor

    Science.gov (United States)

    2016-06-14

    device scaling within the electrodeless insulator dielectrophoresis geometry through the application of highly Fig. 1: Biomarker enrichment in...a competitive electrochemical detection system that is free of wash -steps and enables the real-time monitoring of adenosine triphosphate (ATP) over...methodology for quantifying the region of interest by using a statistical description of particle distribution across the device geometry to determine

  4. An Anthropologist Looks at Malaria | Tobias | South African Medical ...

    African Journals Online (AJOL)

    Also, malaria may be associated with the lower levels of ATP (adenosine triphosphate) in the red blood cells of Blacks. Man's cultural evolution and especially the adoption of agriculture - may have played a big part in the establishment of areas of malarial hyperendemicity. Thus, indirectly, malaria may have helped the early ...

  5. Persistent pain after mastectomy with reconstruction.

    LENUS (Irish Health Repository)

    Hickey, Oonagh T

    2011-09-01

    To determine the prevalence of persistent postsurgical pain (PPSP) and its influence on functional status, and to examine associations between PPSP and single nucleotide polymorphisms of the catechol-O-methyltransferase (COMT) gene and the guanosine triphosphate cyclohydrolase 1 (GCH1) gene following mastectomy and reconstruction.

  6. Author Details

    African Journals Online (AJOL)

    Abstract PDF · Vol 8, No 2 (1994) - Articles Flow injection determinations of glucose and adenosine-5'¬-triphosphate (ATP) based on a packed-bed glucokinase reactor. Abstract PDF · Vol 7, No 2 (1993) - Articles Flow injection spectrophotometric and amperometric determinations of ammonia with glutamate dehydrogenase ...

  7. Dinucleoside polyphosphate NAD analogs as potential NMN adenylyltransferase inhibitors. Synthesis and biological evaluation.

    Science.gov (United States)

    Franchetti, P; Cappellacci, L; Pasqualini, M; Grifantini, M; Lorenzi, T; Raffaelli, N; Magni, G

    2003-01-01

    Two dinucleoside polyphosphate NAD analogs, P1-(adenosine-5')-P3-(nicotinamide riboside-5')triphosphate (Np3A, 1) and P1-(adenosine-5')-P4-(nicotinamide riboside-5')tetraphosphate (Np4A, 2), were synthesized and tested as inhibitors of both microbial and human recombinant NMN adenylyltransferase. Compounds 1 and 2 proved to be selective inhibitors of microbial enzymes.

  8. (Vigna unguiculata (L.) Walp)

    African Journals Online (AJOL)

    komla

    dehydrogenase and phosphoglucoisomerase (glucose- 1 -phosphate). A total of 1 1 0 different alleles were observed; .... 40 mg glucose, 30 mg adenosine diphosphate. (ADP), 7.5 mg nicotinamide adenine dinucleotide ... 100 mg MgClZ, 15 mg adenosine triphosphate. West African Journal of Applied Ecology, v01. 4, 2003 ...

  9. Animation Model to Conceptualize ATP Generation: A Mitochondrial Oxidative Phosphorylation

    Science.gov (United States)

    Jena, Ananta Kumar

    2015-01-01

    Adenosine triphosphate (ATP) is the molecular unit of intracellular energy and it is the product of oxidative phosphorylation of cellular respiration uses in cellular processes. The study explores the growth of the misconception levels amongst the learners and evaluates the effectiveness of animation model over traditional methods. The data…

  10. Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinofuranosylcytosine in leukemia and solid tumor cell lines.

    NARCIS (Netherlands)

    Bergman, A.M.; Kuiper, C.M.; Voorn, D.A.; Comijn, E.M.; Myhren, F.; Sandvold, ML; Hendriks, H.R.; Peters, G.J.

    2004-01-01

    1-beta-D-arabinofuranosylcytosine (ara-C) is a deoxycytidine analog with activity in leukemia, which requires phosphorylation by deoxycytidine kinase (dCK) to allow formation of its active phosphate 1-beta-D-arabinofuranosylcytosine triphosphate, but can be deaminated by deoxycytidine deaminase.

  11. Tetrahydrobiopterin (Bh4) deficiencies: diagnosis and treatment

    OpenAIRE

    Mercedes Martínez-Pardo

    2014-01-01

    Tetrahydrobiopterin (BH4) is an enzyme cofactor of phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase. Three different enzymes affect BH4 synthesis: guanosine triphosphate cyclohydrolase I (GTP CH 1), 6-piruvoil tetrahydropterin synthase (PTPS), sepiapterin reductase (SR) and dihydropteridine reductase (DHPR) affects regeneration. BH4 deficiency causes defects in monoaminergic neurotransmitters synthesis and can be or not associated with hyperphenylalaninemia (HPA)....

  12. The peroxisomal ABC transporter family

    NARCIS (Netherlands)

    Wanders, Ronald J. A.; Visser, Wouter F.; van Roermund, Carlo W. T.; Kemp, Stephan; Waterham, Hans R.

    2007-01-01

    This review describes the current state of knowledge about the ABCD family of peroxisomal half adenosine-triphosphate-binding cassette (ABC) transporters. ABCDs are predicted to be present in a variety of eukaryotic organisms, although at present, only ABCDs in the yeast Saccharomyces cerevisiae,

  13. Homogeneous electrochemical aptamer-based ATP assay with signal amplification by exonuclease III assisted target recycling.

    Science.gov (United States)

    Liu, Shufeng; Wang, Ying; Zhang, Chengxin; Lin, Ying; Li, Feng

    2013-03-21

    A novel and homogeneous electrochemical aptamer-based adenosine triphosphate (ATP) assay was demonstrated with signal amplification by exonuclease III-assisted target recycling. A superior detection limit of 1 nM toward ATP with an excellent selectivity could be achieved.

  14. An experimental-computer modeling study of inorganic phosphates surface adsorption on hydroxyapatite particles.

    Science.gov (United States)

    Rivas, Manuel; Casanovas, Jordi; del Valle, Luis J; Bertran, Oscar; Revilla-López, Guillermo; Turon, Pau; Puiggalí, Jordi; Alemán, Carlos

    2015-06-07

    The adsorption of orthophosphate, pyrophosphate, triphosphate and a trisphosphonate onto hydroxyapatite has been examined using experiments and quantum mechanical calculations. Adsorption studies with FTIR and X-ray photoelectron spectroscopies have been performed considering both crystalline hydroxyapatite (HAp) and amorphous calcium phosphate particles, which were specifically prepared and characterized for this purpose. Density functional theory (DFT) calculations have been carried out considering the (100) and (001) surfaces of HAp, which were represented using 1 × 2 × 2 and 3 × 3 × 1 slab models, respectively. The adsorption of phosphate onto the two crystallographic surfaces is very much favored from an energetic point of view, which is fully consistent with current interpretations of the HAp growing process. The structures calculated for the adsorption of pyrophosphate and triphosphate evidence that this process is easier for the latter than for the former. Thus, the adsorption of pyrophosphate is severely limited by the surface geometry while the flexibility of triphosphate allows transforming repulsive electrostatic interactions into molecular strain. On the other hand, calculations predict that the trisphosphonate only adsorbs onto the (001) surface of HAp. Theoretical predictions are fully consistent with experimental data. Thus, comparison of DFT results and spectroscopic data suggests that the experimental conditions used to prepare HAp particles promote the predominance of the (100) surface. Accordingly, experimental identification of the adsorption of trisphosphonate onto such crystalline particles is unclear while the adsorption of pyrophosphate and triphosphate is clearly observed.

  15. Improved methods for counting bacteria in physiological fluids

    Science.gov (United States)

    Picciolo, G. L.

    1974-01-01

    Bacterial population detection is based on detection of adenosine triphosphate (ATP), chemical present in all living matter. Amount of ATP in sample, after chemically removing all nonbacterial ATP, is directly related to bacterial population. Sensitivity is improved by concentration step; specificity is improved by lowering pH of solution.

  16. Method of detecting and counting bacteria in body fluids

    Science.gov (United States)

    Chappelle, E. W.; Picciolo, G. L. (Inventor)

    1973-01-01

    A novel method is reported for determining bacterial levels in urine samples, which method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of non-bacterial ATP. After the removal of non-bacterial ATP, the bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay using an enzyme obtained from the firefly.

  17. 31P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi

    DEFF Research Database (Denmark)

    Rasmussen, N.; Lloyd, D.C.; Ratcliffe, R.G.

    2000-01-01

    from alpha, beta and gamma phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with this method. The (3)1P NMR...

  18. v V V V V v

    Indian Academy of Sciences (India)

    Il~~ idea. RNA virq$es were known toc~l'i'Ythe. RNA-dependent RNA polymerase enzyme that is essential to make additional copies of the geno- me. A hunt for the virion polymerase was suc- cessful. An enzyme activity that used an RNA template and deoxyribonucleotide triphosphates to synthesize DNA was identified ...

  19. Bioluminometric assay of ATP in mouse brain

    Indian Academy of Sciences (India)

    Firefly luciferase bioluminescence (FLB) is a highly sensitive and specific method for the analysis of adenosine-5-triphosphate (ATP) in biological samples. Earlier attempts to modify the FLB test for enhanced sensitivity have been typically based on in vitro cell systems. This study reports an optimized FLB procedure for the ...

  20. Sympathetic vasoconstriction takes an unexpected pannexin detour

    DEFF Research Database (Denmark)

    Schak Nielsen, Morten

    2015-01-01

    channels and the subsequent release of adenosine triphosphate that promotes contraction in an autocrine and paracrine manner. This elaborate mechanism may function as a point of intercept for other signaling pathways-for example, in relation to the phenomenon "functional sympatholysis," in which exercise...

  1. Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon

    NARCIS (Netherlands)

    Rep, M; van Dijl, J M; Suda, K; Schatz, G; Grivell, L A; Suzuki, C K

    1996-01-01

    Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein

  2. Physico-chemical studies of MgNa3P3O10·12H2O

    Directory of Open Access Journals (Sweden)

    Fahim I.

    2013-09-01

    Full Text Available A magnesium-sodium triphosphate MgNa3P3O10·12H2O already known has been prepared by the method of ion exchange resin of aqueous chemistry and studied by X-Ray diffraction, thermal analyses (TGA-DTA, differential scanning calorimetry (DSC and infrared spectrometry which show the characteristic bands of a triphosphate P3O. The results of differential thermal analysis, X-Ray powder diffraction and IR spectra of the compound heated at different temperatures showed that, after dehydration, MgNa3P3O10·12H2O decomposes into an amorphous compound, then it crystallizes at 600 ° C in order to give the anhydrous triphosphate MgNa3P3O10. MgNa3P3O10 is stable until its melting point at 612 ° C. Two different methods Ozawa and KAS have been selected in studying the kinetics of thermal behavior of the triphosphate P3O10 for the first time. The kinetic and thermodynamic characteristics of the dehydration of MgNa3P3O10·12H2O and the thermal phenomena accompanying this dehydration were determinated and discussed on the basis of the proposed crystalline structure.

  3. Effect of N, P and K Humates on Dry Matter of Zea mays and Soil

    African Journals Online (AJOL)

    Sharjeel Ahmad

    consisting urea, triple superphosphate (TSP) and mono triphosphate (MOP) amended with or without. HA, FA, acidified HA and FA and humin significantly increased the dry matter of the test crop with significant retention of soil exchangeable ammonium. However, only the complete fertilizer with and without HA and humin ...

  4. Polymerase synthesis of oligonucleotides containing a single chemically modified nucleobase for site-specific redox labelling

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Cahová, Hana; Plucnara, Medard; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2013-01-01

    Roč. 49, č. 41 (2013), s. 4652-4654 ISSN 1359-7345 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : cross-coupling reaction s * DNA -protein interactions * nucleoside triphosphates * enzymatic incorporation Subject RIV: CC - Organic Chemistry Impact factor: 6.718, year: 2013

  5. ATP induced vasodilatation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins and adenosine

    DEFF Research Database (Denmark)

    Mortensen, Stefan P; Gonzalez-Alonso, Jose; Bune, Laurids

    2009-01-01

    Plasma adenosine-5'-triphosphate (ATP) is thought to contribute to the local regulation of skeletal muscle blood flow. Intravascular ATP infusion can induce profound limb muscle vasodilatation, but the purinergic receptors and downstream signals involved in this response remain unclear. This study...

  6. Contribution to the study of isotopic exchange of hydrogen in purine derivatives

    International Nuclear Information System (INIS)

    Valenta, V.

    1977-01-01

    The dependence was studied of the rate of hydrogen isotopic exchange in purine derivatives in aqueous media. The effect of pH and of temperature was investigated in adenosine, adenosine monophosphate and adenosine triphosphate, and of pH in guanosine, guanosine monophosphate and guanosine triphosphate. The isotopic exchange of hydrogen was studied in the system hexamethyl phosphotriamide - [ 3 H]water - purine derivative (adenine, adenosine, adenosine monophosphate). The reaction mechanism of hydrogen isotopic exchange is discussed. It may be inferred from the measured value of the kinetic isotopic effect. The ''reverse'' isotopic exchange of hydrogen was investigated in [8- 3 H]adenosine, [8- 3 H]adenosine-5'-monophosphate, and [8- 3 H]adenosine-5'-triphosphate. The possible use was verified of the hydrogen isotopic exchange in the system tritium gas - water - purine derivative, catalyzed by metal catalysts, for the preparation of purine derivatives labelled with tritium in position 8. The effect of pH of, the catalyst type and of quantity was studied. Adenosine-5'-triphosphate with a molar activity of 860 GBq/mmol was prepared using carrier-free tritium gas. (author)

  7. Tris stimulated ecdysteroid secretion via Ca2+ messenger system in the prothoracic glands of Locusta migratoria

    Czech Academy of Sciences Publication Activity Database

    Neuwirth, Aleš; Kodrík, Dalibor; Birkenbeil, H.; Sehnal, František

    2005-01-01

    Roč. 30, - (2005), s. 270-277 ISSN 0307-6962 Grant - others:Deutsch Gesellschaft fur Luft-und Raumfahrt(DE) TSR-072-97 Institutional research plan: CEZ:AV0Z50070508 Keywords : Calcium channels * ecdysteroids * inositol triphosphate Subject RIV: ED - Physiology Impact factor: 1.221, year: 2005

  8. Absence of 633-nm laser irradiation-induced effects on glucose phosphorylation by hexokinase

    NARCIS (Netherlands)

    Heger, Michal; Heemskerk, Anthonius A. M.; van der Zwan, Gert

    2010-01-01

    In a paper by Amat et al. (Modification of the intrinsic fluorescence and biochemical behavior of adenosine triphosphate ATP after irradiation with visible and near-infrared laser light, J. Photochem. Photobiol. B 81 (2005) 26-32) it was shown that the conversion of glucose to glucose-6-phosphate by

  9. Identification and Characterization of the Nuclear Isoform of Drosophila melanogaster CTP:Phosphocholine Cytidylyltransferase

    Science.gov (United States)

    CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the conversion of phosphocholine and cytidine 5'-triphosphate (CTP) to CDP-choline for the eventual synthesis of phosphatidylcholine (PC). The enzyme is regulated by reversible association with cellular membranes, with the rate of catalysis in...

  10. Intracellular phosphorylation of benzyladenosine is related to apoptosis induction in tobacco BY-2 cells

    Czech Academy of Sciences Publication Activity Database

    Mlejnek, P.; Doležel, Jaroslav; Procházka, S.

    2003-01-01

    Roč. 26, č. 10 (2003), s. 1723-1735 ISSN 0140-7791 R&D Projects: GA MŠk LN00A081 Institutional research plan: CEZ:AV0Z5038910 Keywords : adenosine 5'-triphosphate * cytokinin * N6-benzyladenine 9-riboside Subject RIV: ED - Physiology Impact factor: 3.613, year: 2003

  11. Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

    NARCIS (Netherlands)

    Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

    1998-01-01

    Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

  12. ATP-sensitive potassium channels in isolated rat aorta during physiologic, hypoxic, and low-glucose conditions

    NARCIS (Netherlands)

    Hüsken, B. C.; Pfaffendorf, M.; van Zwieten, P. A.

    1997-01-01

    In arterial smooth muscle, adenosine triphosphate (ATP)-sensitive potassium channels are the targets of a variety of synthetic and endogenous vasodilators. In this study, we evaluated the influence of glibenclamide, an ATP-sensitive K(+)-channel blocker, on various vasodilator responses, including

  13. Detergent inhibited, heat labile nucleoside triphosphatase in cores of avian myeloblastosis virus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis. This t...

  14. Synthesis, bioanalysis and pharmacology of nucleoside and nucleotide analogs

    NARCIS (Netherlands)

    Jansen, R.S.

    2009-01-01

    Nucleoside analogs are an important class of drugs in anticancer and antiviral therapy. The compounds are, however, only active after intracellular conversion to their mono-, di- and triphosphate nucleotide form. In this thesis the development of sensitive liquid chromatography coupled to tandem

  15. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA

    Czech Academy of Sciences Publication Activity Database

    Ikonen, Satu; Macíčková-Cahová, Hana; Pohl, Radek; Šanda, Miloslav; Hocek, Michal

    2010-01-01

    Roč. 8, č. 5 (2010), s. 1194-1201 ISSN 1477-0520 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : steroids * nucleosides * nucleoside triphosphates * DNA polymerase Subject RIV: CC - Organic Chemistry Impact factor: 3.451, year: 2010

  16. Labeling of thymine with 99m technetium: a suggestion of a chemical model

    International Nuclear Information System (INIS)

    Gutfilen, Bianca; Silva, Claudia Ribeiro da; Bernardo Filho, Mario; Ribeiro, Barbara Luzia Almeida; Mattos, Maura Ferreira

    1996-01-01

    Successful targeting of diagnose but also to stage cancer. It has been shown that certain tumor cells are permeable to low level of exogenous adenosine-diphosphate and adenosine-triphosphate nucleotides, that are incorporated into intracellular pools. We present the labeling of a nucleotide precursor, a base, thymine technetium-99m ( 99m Tc). (author)

  17. Labeling of thymine with {sup 99m} technetium: a suggestion of a chemical model

    Energy Technology Data Exchange (ETDEWEB)

    Gutfilen, Bianca; Silva, Claudia Ribeiro da; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria; Ribeiro, Barbara Luzia Almeida [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Mattos, Maura Ferreira [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Quimica

    1996-03-01

    Successful targeting of diagnose but also to stage cancer. It has been shown that certain tumor cells are permeable to low level of exogenous adenosine-diphosphate and adenosine-triphosphate nucleotides, that are incorporated into intracellular pools. We present the labeling of a nucleotide precursor, a base, thymine technetium-99m ({sup 99m} Tc). (author)

  18. Free-energy carriers in human cultured muscle cells

    NARCIS (Netherlands)

    Bolhuis, P. A.; de Zwart, H. J.; Ponne, N. J.; de Jong, J. M.

    1985-01-01

    Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media

  19. AcEST: DK952901 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Definition tr|A8A8G8|A8A8G8_IGNH4 Anaerobic ribonucleoside-triphosphate reductase OS=Ignicoccus hospital...te reductase OS=Ignicoccus hospitalis (strain KIN4/I / DSM 18386 / JCM 14125) GN=Igni_0036 PE=4 SV=1 Length

  20. Benzofurazane as a New Redox Label for Electrochemical Detection of DNA: Towards Multipotential Redox Coding of DNA Bases

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Plucnara, Medard; Vidláková, Pavlína; Pohl, Radek; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2013-01-01

    Roč. 19, č. 38 (2013), s. 12720-12731 ISSN 0947-6539 R&D Projects: GA ČR GBP206/12/G151; GA AV ČR(CZ) IAA400040901 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : DNA polymerase * electrochemistry * nucleoside triphosphates * sequencing * voltammetry Subject RIV: CC - Organic Chemistry Impact factor: 5.696, year: 2013

  1. Application of Group-Based QSAR and Molecular Docking in the ...

    African Journals Online (AJOL)

    Conclusion: The generated models provide a site-specific insight into the structural requirements for IGF-1R inhibition which can be used to design and develop potent inhibitors. Keywords: Insulin-like growth factor 1 (IGF-1) receptor, Quantitative structure-activity relationship, Adenosine triphosphate, Competitive inhibitors, ...

  2. Fulltext PDF

    Indian Academy of Sciences (India)

    2010-12-06

    triphosphate (IP3) and diacylglycerol (DG). Further, IP3 promotes the release of Ca2+ from endoplas- mic reticulum into plasma by binding it with its type-1 IP3 receptor, which was anchored in the membrane of endo- plasmic reticulum.

  3. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  4. The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.

    Science.gov (United States)

    Mauney, Christopher H; Rogers, LeAnn C; Harris, Reuben S; Daniel, Larry W; Devarie-Baez, Nelmi O; Wu, Hanzhi; Furdui, Cristina M; Poole, Leslie B; Perrino, Fred W; Hollis, Thomas

    2017-12-01

    Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond "redox switch" that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.

  5. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  6. The stochastic chemomechanics of the F(1)-ATPase molecular motor.

    Science.gov (United States)

    Gaspard, P; Gerritsma, E

    2007-08-21

    We report a theoretical study of the F(1)-ATPase molecular rotary motor experimentally studied by R. Yasuda, H. Noji, M. Yoshida, K. Kinosita Jr., H. Itoh [Nature 410 (2001) 898]. The motor is modeled as a stochastic process for the angle of its shaft and the chemical state of its catalytic sites. The stochastic process is ruled by six coupled Fokker-Planck equations for the biased diffusion of the angle and the random jumps between the chemical states. The model reproduces the experimental observations that the motor proceeds by substeps and the rotation rate saturates at high concentrations of adenosine triphosphate or at low values of the friction coefficient. Moreover, predictions are made about the dependence of the rotation rate on temperature, and about the behavior of the F(1) motor under the effect of an external torque, especially, in the regime of synthesis of adenosine triphosphate.

  7. Deprotonated imidodiphosphate in AMPPNP-containing protein structures

    International Nuclear Information System (INIS)

    Dauter, Miroslawa; Dauter, Zbigniew

    2011-01-01

    In certain AMPPNP-containing protein structures, the nitrogen bridging the two terminal phosphate groups can be deprotonated. Many different proteins utilize the chemical energy provided by the cofactor adenosine triphosphate (ATP) for their proper function. A number of structures in the Protein Data Bank (PDB) contain adenosine 5′-(β,γ-imido)triphosphate (AMPPNP), a nonhydrolysable analog of ATP in which the bridging O atom between the two terminal phosphate groups is substituted by the imido function. Under mild conditions imides do not have acidic properties and thus the imide nitrogen should be protonated. However, an analysis of protein structures containing AMPPNP reveals that the imide group is deprotonated in certain complexes if the negative charges of the phosphate moieties in AMPPNP are in part neutralized by coordinating divalent metals or a guanidinium group of an arginine

  8. Counteraction by MutT protein of transcriptional errors caused by oxidative damage.

    Science.gov (United States)

    Taddei, F; Hayakawa, H; Bouton, M; Cirinesi, A; Matic, I; Sekiguchi, M; Radman, M

    1997-10-03

    Oxidized guanine (8-oxo-7,8-dihydroguanine; 8-oxo-G) is a potent mutagen because of its ambiguous pairing with cytosine and adenine. The Escherichia coli MutT protein specifically hydrolyzes both 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-rGTP), which are otherwise incorporated in DNA and RNA opposite template A. In vivo, this cleaning of the nucleotide pools decreases both DNA replication and transcription errors. The effect of mutT mutation on transcription fidelity was shown to depend on oxidative metabolism. Such control of transcriptional fidelity by the ubiquitous MutT function has implications for evolution of RNA-based life, phenotypic expression, adaptive mutagenesis, and functional maintenance of nondividing cells.

  9. Effects of overexpression and antisense RNA expression of Orf17, a MutT-type enzyme.

    Science.gov (United States)

    Hori, Mika; Asanuma, Taketoshi; Inanami, Osamu; Kuwabara, Mikinori; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2006-06-01

    The Escherichia coli Orf17 (NtpA, NudB) protein, a MutT-type enzyme, hydrolyzes oxidized deoxyribonucleotides, including 8-hydroxy-2'-deoxyadenosine 5'-triphosphate and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, in vitro. To examine its in vivo role(s) in bacteria, plasmid DNAs containing the orf17 gene in the sense and antisense orientations were introduced. When the Orf17 protein was overexpressed in mutT cells, the rpoB mutant frequency was decreased. On the other hand, similar effects were not observed when Orf17 was overexpressed in wild type and orf135 cells. Expression of the antisense RNA of the orf17 gene did not produce an obvious phenotype, such as increased mutant frequency and resistance to ionizing radiation. These results suggest that the role of the Orf17 protein is to back up the MutT function, and to assist in the elimination of 8-hydroxy-2'-deoxyguanosine nucleotides.

  10. Advantages and limitations of experimental techniques used to measure cardiac energy metabolism.

    Science.gov (United States)

    Lopaschuk, G D

    1997-01-01

    The heart requires a constant supply of energy to sustain contractile function, which is supplied by hydrolysis of adenosine triphosphate derived primarily from the metabolism of fatty acids and carbohydrates. Understanding how production of adenosine triphosphate is regulated in the heart is critical to an understanding of how alterations in energy metabolism contribute to the severity of cardiac disease. A number of techniques can be used to measure energy metabolism in the heart. They include biochemical measurement of metabolites and enzymes of intermediary metabolism, measurement of arteriovenous differences in carbon substrate extraction by the heart, measurement of high-energy phosphates with 31P nuclear magnetic resonance, measurement of the rate of flux through the pathways of intermediary metabolism with 14C- and 3H-labeled carbon substrates, measurement of tricarboxylic acid cycle activity with 13C nuclear magnetic resonance, and measurement of glucose uptake and oxidative metabolism with positron emission tomography. Each of these techniques has advantages and limitations.

  11. The role of energy in the emergence of biology from chemistry.

    Science.gov (United States)

    Dibrova, Daria V; Chudetsky, Michail Y; Galperin, Michael Y; Koonin, Eugene V; Mulkidjanian, Armen Y

    2012-10-01

    Any scenario of the transition from chemistry to biology should include an "energy module" because life can exist only when supported by energy flow(s). We addressed the problem of primordial energetics by combining physico-chemical considerations with phylogenomic analysis. We propose that the first replicators could use abiotically formed, exceptionally photostable activated cyclic nucleotides both as building blocks and as the main energy source. Nucleoside triphosphates could replace cyclic nucleotides as the principal energy-rich compounds at the stage of the first cells, presumably because the metal chelates of nucleoside triphosphates penetrated membranes much better than the respective metal complexes of nucleoside monophosphates. The ability to exploit natural energy flows for biogenic production of energy-rich molecules could evolve only gradually, after the emergence of sophisticated enzymes and ion-tight membranes. We argue that, in the course of evolution, sodium-dependent membrane energetics preceded the proton-based energetics which evolved independently in bacteria and archaea.

  12. Phosphatidylinositol 4-phosphate 5-kinases in the regulation of T cell activation

    Directory of Open Access Journals (Sweden)

    Loretta eTuosto

    2016-05-01

    Full Text Available Phosphatidylinositol 4,5-biphosphate kinases (PIP5K are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2. PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen presenting cells (APC, spatial organization of the immunological synapse (IS, and costimulation. Moreover, PIP2 serves also as a precursor for the second messengers inositol triphosphate (IP3, diacylglycerol (DAG and phosphatidylinositol 3,4,5-triphosphate (PIP3, which are essential for the activation of signalling pathways regulating cytokine production, cell cycle progression, survival, metabolism and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation.

  13. Using a Genome-Scale Metabolic Network Model to Elucidate the Mechanism of Chloroquine Action in Plasmodium falciparum

    Science.gov (United States)

    2017-03-22

    a critical gene.2.7. Identifying genes underlying chloroquine-induced DNA synthesis inhibition Chloroquine administration inhibits DNA replication in...Drug Resistance 7 (2017) 138e1461442008). Our proposed mechanism, which is summarized in Fig. 5, suggests that administration of chloroquine has two...diphosphate; dTTP, deoxythymidine triphosphate; G3P, glycer- oin; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADPþ, nicotin

  14. Synthesis of acetylene linked double-nucleobase nucleos(t)ide building blocks and polymerase construction of DNA containing cytosines in the major groove

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Pohl, Radek; Hocek, Michal

    2011-01-01

    Roč. 76, č. 9 (2011), s. 3457-3462 ISSN 0022-3263 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside triphosphates * functionalized DNA * cross-coupling reactions * nucleic-acids Subject RIV: CC - Organic Chemistry Impact factor: 4.450, year: 2011

  15. Rapid methods for detection of bacteria

    DEFF Research Database (Denmark)

    Corfitzen, Charlotte B.; Andersen, B.Ø.; Miller, M.

    2006-01-01

    Traditional methods for detection of bacteria in drinking water e.g. Heterotrophic Plate Counts (HPC) or Most Probable Number (MNP) take 48-72 hours to give the result. New rapid methods for detection of bacteria are needed to protect the consumers against contaminations. Two rapid methods: Measu......: Measurements of Adosine Triphosphate and BactiQuantTM have shown promising results as new monitoring tools, which gives the result within minutes/hours....

  16. Age-associated metabolic and morphologic changes in mitochondria of individual mouse and hamster oocytes.

    Directory of Open Access Journals (Sweden)

    Fatma Simsek-Duran

    Full Text Available BACKGROUND: In human oocytes, as in other mammalian ova, there is a significant variation in the pregnancy potential, with approximately 20% of oocyte-sperm meetings resulting in pregnancies. This frequency of successful fertilization decreases as the oocytes age. This low proportion of fruitful couplings appears to be influenced by changes in mitochondrial structure and function. In this study, we have examined mitochondrial biogenesis in both hamster (Mesocricetus auratus and mouse (Mus musculus ova as models for understanding the effects of aging on mitochondrial structure and energy production within the mammalian oocyte. METHODOLOGY/PRINCIPAL FINDINGS: Individual metaphase II oocytes from a total of 25 young and old mice and hamsters were collected from ovarian follicles after hormone stimulation and prepared for biochemical or structural analysis. Adenosine triphosphate levels and mitochondrial DNA number were determined within individual oocytes from young and old animals. In aged hamsters, oocyte adenosine triphosphate levels and mitochondrial DNA molecules were reduced 35.4% and 51.8%, respectively. Reductions of 38.4% and 44% in adenosine triphosphate and mitochondrial genomes, respectively, were also seen in aged mouse oocytes. Transmission electron microscopic (TEM analysis showed that aged rodent oocytes had significant alterations in mitochondrial and cytoplasmic lamellae structure. CONCLUSIONS/SIGNIFICANCE: In both mice and hamsters, decreased adenosine triphosphate in aged oocytes is correlated with a similar decrease in mtDNA molecules and number of mitochondria. Mitochondria in mice and hamsters undergo significant morphological change with aging including mitochondrial vacuolization, cristae alterations, and changes in cytoplasmic lamellae.

  17. Condiciones de limpieza de superficies próximas al paciente en una unidad de terapia intensiva

    OpenAIRE

    Ferreira, Adriano Menis; Andrade, Denise de; Rigotti, Marcelo Alessandro; Ferreira, Maria Verônica Ferrareze

    2011-01-01

    Surface cleaning is a well-known control procedure against the dissemination of microorganisms in the hospital environment. This prospective study, carried out in an intensive care unit over the course of 14 days, describes the cleaning/disinfection conditions of four surfaces near patients. In total, 100 assessments of the surfaces were carried out after they were cleaned. Three methods were used to evaluate cleanliness: a visual inspection, an adenosine triphosphate (ATP) bioluminescence as...

  18. Urinary ATP as an indicator of infection and inflammation of the urinary tract in patients with lower urinary tract symptoms

    OpenAIRE

    Gill, Kiren; Horsley, Harry; Kupelian, Anthony S; Baio, Gianluca; De Iorio, Maria; Sathiananamoorthy, Sanchutha; Khasriya, Rajvinder; Rohn, Jennifer L; Wildman, Scott S.P.; Malone-Lee, James

    2015-01-01

    BACKGROUND:\\ud Adenosine-5'-triphosphate (ATP) is a neurotransmitter and inflammatory cytokine implicated in the pathophysiology of lower urinary tract disease. ATP additionally reflects microbial biomass thus has potential as a surrogate marker of urinary tract infection (UTI). The optimum clinical sampling method for ATP urinalysis has not been established. We tested the potential of urinary ATP in the assessment of lower urinary tract symptoms, infection and inflammation, and validated sam...

  19. Neuron-glia interactions in glutamatergic neurotransmission

    DEFF Research Database (Denmark)

    Schousboe, A; Sickmann, H M; Bak, Lasse Kristoffer

    2011-01-01

    Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear...... theses processes also has not been fully elucidated. Cultured astrocytes and neurons were utilized to monitor these processes related to glutamatergic neurotransmission. Inhibitors of glycolysis and TCA cycle in combination with pathway-selective substrates were used to study glutamate uptake and release...

  20. A Powerful Mitochondria-Targeted Iron Chelator Affords High Photoprotection against Solar Ultraviolet A Radiation

    OpenAIRE

    Reelfs, Olivier; Abbate, Vincenzo; Hider, Robert C.; Pourzand, Charareh

    2016-01-01

    Mitochondria are the principal destination for labile iron, making these organelles particularly susceptible to oxidative damage on exposure to ultraviolet A (UVA, 320?400 nm), the oxidizing component of sunlight. The labile iron-mediated oxidative damage caused by UVA to mitochondria leads to necrotic cell death via adenosine triphosphate depletion. Therefore, targeted removal of mitochondrial labile iron via highly specific tools from these organelles may be an effective approach to protect...

  1. [Thiamine and its derivatives in the regulation of cell metabolism].

    Science.gov (United States)

    Tylicki, Adam; Siemieniuk, Magdalena

    2011-07-06

    For over 70 years thiamine (vitamin B1) has aroused the interest of biologists, biochemists and medical doctors because of its multilateral participation in key biochemical and physiological processes. The thiamine molecule is composed of pyrimidine and thiazole rings which are linked by a methylene bridge. It is synthesized by microorganisms, fungi and plants, whereas animals and humans have to obtain it from food. There are several known forms of vitamin B1 inside cells: free thiamine, three phosphate esters (mono-, di-, and triphosphate), and the recently found adenosine thiamine triphosphate. Thiamine has a dual, coenzymatic and non-coenzymatic role. First of all, it is a precursor of thiamin diphosphate, which is a coenzyme for over 20 characterized enzymes which are involved in cell bioenergetic processes leading to the synthesis of ATP. Moreover, these enzymes take part in the biosynthesis of pentose (required for the synthesis of nucleotides), amino acids and other organic compounds of cell metabolism. On the other hand, recent discoveries show the non-coenzymatic role of thiamine derivatives in the process of regulation of gene expression (riboswitches in microorganisms and plants), the stress response, and perhaps so far unknown signal transduction pathways associated with adverse environmental conditions, or transduction of nerve signals with participation of thiamine triphosphate and adenosine thiamine triphosphate. From the clinical point of view thiamine deficiency is related to beri-beri, Parkinson disease, Alzheimer disease, Wernicke-Korsakoff syndrome and other pathologies of the nervous system, and it is successfully applied in medical practice. On the other hand, identifying new synthetic analogues of thiamine which could be used as cytostatics, herbicides or agents preventing deficiency of vitamin B1 is currently the major goal of the research. In this paper we present the current state of knowledge of thiamine and its derivatives, indicating

  2. Effects of Monotypic and Binary Mixtures of Metal Oxide Nanoparticles on Microbial Growth in Sandy Soil Collected from Artificial Recharge Sites

    OpenAIRE

    Ko, Kyung-Seok; Ha, Kyoochul; Kong, In Chul

    2015-01-01

    The potential effects of monotypic and binary metal oxide nanoparticles (NPs, ZnO, NiO, Co3O4 and TiO2) on microbial growth were evaluated in sandy soil collected from artificial recharge sites. Microbial growth was assessed based on adenosine triphosphate (ATP) content, dehydrogenase activity (DHA), and viable cell counts (VCC). Microbial growth based on ATP content and VCC showed considerable differences depending on NP type and concentration, whereas DHA did not significantly change. In ge...

  3. Practical Considerations For Developing Nucleoside Reverse Transcriptase Inhibitors

    OpenAIRE

    Hurwitz, Selwyn J.; Schinazi, Raymond F.

    2012-01-01

    Nucleoside reverse transcriptase inhibitors (NRTI) remain a cornerstone of current antiretroviral regimens in combinations usually with a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), or an integrase inhibitor (INI). The antiretroviral efficacy and relative safety of current NRTI results from a tight and relatively specific binding of their phosphorylated nucleoside triphosphates (NRTI-TP) with the HIV-1 reverse transcriptase which is ess...

  4. Adenosine as an endogenous immunoregulator in cancer pathogenesis: where to go?

    OpenAIRE

    Kumar, V.

    2012-01-01

    Cancer is a chronic disease and its pathogenesis is well correlated with infection and inflammation. Adenosine is a purine nucleoside, which is produced under metabolic stress like hypoxic conditions. Acute or chronic inflammatory conditions lead to the release of precursor adenine nucleotides (adenosine triphosphate (ATP), adenosien diphosphate (ADP) and adenosine monophosphate (AMP)) from cells, which are extracellularly catabolized into adenosine by extracellular ectonucleotidases, i.e., C...

  5. Cultured astrocytes do not release adenosine during hypoxic conditions

    OpenAIRE

    Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

    2011-01-01

    Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adeno...

  6. Purine receptors in the trachea: is there a receptor for ATP?

    OpenAIRE

    Christie, J.; Satchell, D. G.

    1980-01-01

    In guinea-pig trachea adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-phosphate (AMP), adenosine and adenine were similarly potent in causing relaxation of the smooth muscle. This is in contrast to gut where ATP and ADP are 30 times more potent than adenosine. Studies with dipyridamole suggest that in trachea, as in gut, nucleotides are rapidly metabolized to adenosine. A polyphosphate modified analogue of ATP, the alpha,beta-methylene isostere, which resists deg...

  7. The effect of adenyl compounds on the rat heart.

    OpenAIRE

    Burnstock, G.; Meghji, P.

    1983-01-01

    1 The effects of adenyl compounds were examined on the rat atrium and ventricle. 2 Adenosine, adenosine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-triphosphate (ATP) and beta, gamma-methylene ATP (APPCP) produced negative inotropic effects on the rat atrium. These inhibitory effects were antagonized by 8-phenyltheophylline (8-PT), a P1-purinoceptor antagonist, and potentiated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, but were not affected...

  8. Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

    OpenAIRE

    Nakamura, S; Shimooku, K; Akisue, T; Jinnai, H; Hitomi, T; Kiyohara, Y; Ogino, C; Yoshida, K; Nishizuka, Y

    1995-01-01

    Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and th...

  9. A modified approach for rapid determination of the quality of industrially produced P-33 orthophosphoric acid preparations

    International Nuclear Information System (INIS)

    Abdukayumov, M. N.; Chistyakov, P. G.; Shilin, E. A.

    2002-01-01

    In this work we have developed an approach to rapidly estimate the quality of P-33 labeled Orthophosphoric Acid (carrier free) in industrial production. The method was created to determine the values of specific radioactivity for both Orthophosphoric acid and gamma-Adenosine 5'-Triphosphate (ATP) by means of enzymatic ATP synthesis in equal molar ratio conditions for two main substrates (radioactive Orthophosphoric acid and ADP) with following chromatography of the reaction mixture on the PEI-cellulose micro column

  10. Isolated HeLa cell nuclei synthesize meaningful DNA.

    OpenAIRE

    Kristensen, T; Prydz, H

    1985-01-01

    DNA replicated at the beginning of S phase was labelled by incubating nuclei isolated from cells arrested at the G/S border with radioactive deoxyribonucleoside triphosphate in a reaction mixture sustaining DNA synthesis. By hybridization against ribosomal RNA bound to nitrocellulose, the fraction of the labelled DNA which was complementary to rRNA could be quantified, and the stability of the RNA-DNA hybrids could be estimated by sequential elution of DNA at increasing temperatures. The resu...

  11. Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-Protein Interactions by F-19 NMR Spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Olszewska, Agata; Pohl, Radek; Hocek, Michal

    2017-01-01

    Roč. 82, č. 21 (2017), s. 11431-11439 ISSN 0022-3263 R&D Projects: GA ČR GBP206/12/G151 Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : RNA secondary structures * cross-linking * 2'-deoxyribonudeoside triphosphates Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 4.849, year: 2016

  12. Nuclear DNA synthesis in yeast and the effect of irradiation

    International Nuclear Information System (INIS)

    Resnick, M.A.; Martin, P.

    1977-01-01

    The synthesis of DNA could be measured in yeast by following the uptake of 5-bromodeoxy-uridine-5'-triphosphate in a mutant that utilizes deoxythymidine-5'-monophosphate; approximately 60 per cent of the DNA was synthesized semi-conservatively before replication stopped. Neither ultraviolet light (U.V.), nor ionizing radiation stimulated repair-type synthesis. Based on the ability to detect small amounts of synthesis, it appeared that fewer than ten bases were synthesized per pyrimidine dimer removed. (author)

  13. Method of detecting and counting bacteria

    Science.gov (United States)

    Picciolo, G. L.; Chappelle, E. W. (Inventor)

    1976-01-01

    An improved method is provided for determining bacterial levels, especially in samples of aqueous physiological fluids. The method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of nonbacterial ATP. The bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay. A concentration technique is included to make the method more sensitive. It is particularly useful where the fluid to be measured contains an unknown or low bacteria count.

  14. Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

    OpenAIRE

    Masker, W E; Dodson, L A; Maupin, M

    1985-01-01

    We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.

  15. Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

    Science.gov (United States)

    Masker, W E; Dodson, L A; Maupin, M

    1985-01-01

    We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested. PMID:3903213

  16. Polymerase synthesis of DNA labelled with benzylidene cyanoacetamide-based fluorescent molecular rotors: fluorescent light-up probes for DNA-binding proteins

    Czech Academy of Sciences Publication Activity Database

    Dziuba, Dmytro; Pohl, Radek; Hocek, Michal

    2015-01-01

    Roč. 51, č. 23 (2015), s. 4880-4882 ISSN 1359-7345 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : enzyme amplification reaction * modified oligonucleotides * nucleoside triphosphates Subject RIV: CC - Organic Chemistry Impact factor: 6.567, year: 2015 http://pubs.rsc.org/en/content/articlepdf/2015/cc/c5cc00530b

  17. Effects of sleep deprivation on sleep homeostasis and restoration during methadone-maintenance: a [31] P MRS brain imaging study

    OpenAIRE

    Trksak, George H.; Jensen, J. Eric; Plante, David T.; Penetar, David M.; Tartarini, Wendy L.; Maywalt, Melissa A.; Brendel, Michael; Dorsey, Cynthia M.; Renshaw, Perry F.; Lukas, Scott E.

    2009-01-01

    Insomnia afflicts many individuals, but particularly those in chronic methadone treatment. Studies examining sleep deprivation (SD) have begun to identify sleep restoration processes involving brain bioenergetics. The technique [31]P magnetic resonance spectroscopy (MRS) can measure brain changes in the high-energy phosphates: alpha-, beta-, and gamma-nucleoside triphosphate (NTP). In the present study, 21 methadone-maintained (MM) and 16 control participants underwent baseline (BL), SD (40 w...

  18. Discovery of External Modulators of the Fe-Fe Hydrogenase Enzyme in Clostridium acetobutylicum

    Science.gov (United States)

    2015-02-01

    challenge in this report is the computational discovery of small molecule inhibitors. There are tens of millions of commercially available small compounds...and Marburg viruses6–9 and an ATP (adenosine triphosphate)–powered secretion system in plague bacteria.10 Two million commercially available...monitored at 15-min intervals throughout the fermentation using gas chromatography. The minimum volume for running our bioreactors is normally 0.5 l. Thus

  19. An untargeted LC-MS investigation of South African children with respiratory chain deficiencies / Leonie Venter

    OpenAIRE

    Venter, Leonie

    2014-01-01

    Mitochondria are the main site of cellular adenosine triphosphate (ATP) generation which is achieved by a series of multi-subunit complexes and electron carriers which together create the oxidative phosphorylation system (OXPHOS). Whenever a defect in any of the numerous mitochondrial pathways occurs it is commonly referred to as a mitochondrial disorder. Mitochondrial disorders are a heterogeneous group of disorders characterised by impaired energy production and include a wide range of defe...

  20. Effects of nucleotides and nucleosides on coagulation

    DEFF Research Database (Denmark)

    Bune, Laurids; Thaning, Pia; Johansson, Pär I

    2010-01-01

    Nucleotides, including ADP, ATP and uridine triphosphate (UTP), are discharged profusely in the circulation during many pathological conditions including sepsis. Sepsis can cause hypotension and systemic activation of the coagulation and fibrinolytic systems in humans, which may cause disseminated...... intravascular coagulation. We investigated whether nucleotide-induced cardiovascular collapse as provoked by systemic infusion of adenosine, ADP, ATP, UTP and nitric oxide affected the haemostatic system as assessed by whole blood thromboelastography (TEG) analysis. Ten pigs received a randomized infusion...

  1. Carbohydrate sources of microfouling material developed on aluminium and stainless steel panels

    Digital Repository Service at National Institute of Oceanography (India)

    Bhosle, N.B.; Sankaran, P.D.; Wagh, A.B.

    was analysed for microfouling biomass (as dry weight andtor adenosine triphosphate), carbohydrate content and composition. l'\\eutral carbohydrates wcrc determineda~allitol acetates in hydrolysatcs of the microfouling samples. With increase in the Imml... in the microfouling material obtained from these substrata. The rnannose/xylosc ratios « 5%), the glucose free weight combined percentage distribution of ribose and fucose (> 12%) as well as arabinose and galactose I> 28%) suggest that plankton, non-woody vascular...

  2. Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples.

    OpenAIRE

    Chen, D; Harke, H R; Dovichi, N J

    1992-01-01

    We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in the original protocol, the sequencing reaction uses modified T7 polymerase with manganese rather than magnesium to produce very uniform incorporation of each dideoxynucleoside. To improve sequencing accuracy, two fluorescently labeled primers are employed in separate sequencing reactions. As an example, one sequencing reaction uses a FAM-labeled primer with dideoxyadenosine triphosphate...

  3. 5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs

    Czech Academy of Sciences Publication Activity Database

    Cahová, Hana; Panattoni, Alessandro; Kielkowski, Pavel; Fanfrlík, Jindřich; Hocek, Michal

    2016-01-01

    Roč. 11, č. 11 (2016), s. 3165-3171 ISSN 1554-8929 R&D Projects: GA ČR GA14-04289S EU Projects: European Commission(XE) 642023 - ClickGene Institutional support: RVO:61388963 Keywords : enzymatic synthesis * 2'-deoxyribonucleoside triphosphates * restriction endonucleases Subject RIV: CC - Organic Chemistry Impact factor: 4.995, year: 2016 http://pubs.acs.org/doi/full/10.1021/acschembio.6b00714

  4. Mesenchymal Stem Cells Induce Expression of CD73 in Human Monocytes In Vitro and in a Swine Model of Myocardial Infarction In Vivo

    OpenAIRE

    Marta Monguió-Tortajada; Marta Monguió-Tortajada; Santiago Roura; Santiago Roura; Santiago Roura; Carolina Gálvez-Montón; Carolina Gálvez-Montón; Marcella Franquesa; Marcella Franquesa; Antoni Bayes-Genis; Antoni Bayes-Genis; Antoni Bayes-Genis; Antoni Bayes-Genis; Francesc E. Borràs; Francesc E. Borràs

    2017-01-01

    The ectoenzymes CD39 and CD73 regulate the purinergic signaling through the hydrolysis of adenosine triphosphate (ATP)/ADP to AMP and to adenosine (Ado), respectively. This shifts the pro-inflammatory milieu induced by extracellular ATP to the anti-inflammatory regulation by Ado. Mesenchymal stem cells (MSCs) have potent immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype aiding tissue repair. In vitro, we observed that human cardiac adipose tiss...

  5. Game of 'Somes: Protein Destruction for Mycobacterium tuberculosis Pathogenesis.

    Science.gov (United States)

    Samanovic, Marie I; Darwin, K Heran

    2016-01-01

    The proteasome system of Mycobacterium tuberculosis is required for causing disease. Proteasomes are multisubunit chambered proteases and, until recently, were only known to participate in adenosine triphosphate (ATP)-dependent proteolysis in bacteria. In this review, we discuss the latest advances in understanding how both ATP-dependent and ATP-independent proteasome-regulated pathways contribute to M. tuberculosis virulence. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. CAP binding proteins associated with the nucleus.

    OpenAIRE

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in in...

  7. Emergence of Fundamental Limits in Spatially Distributed Dynamical Networks and Their Tradeoffs

    Science.gov (United States)

    2017-05-01

    systems. Minimal model of glycolysis . The central role of glycolysis is to consume glucose and produce adenosine triphosphate (ATP), the cells energy...currency. Similar to many other engineered systems whose machinery runs on its own energy product, the glycolysis reaction is autocatalytic. The ATP...the case of the glycolysis pathway, where V > 0 depends on s, parameter q > 0 captures the strength of autocatalysis, and "(, h > 0 capture the

  8. Azidophenyl as a click-transformable redox label of DNA suitable for electrochemical detection of DNA-protein interactions

    Czech Academy of Sciences Publication Activity Database

    Balintová, Jana; Špaček, Jan; Pohl, Radek; Brázdová, Marie; Havran, Luděk; Fojta, Miroslav; Hocek, Michal

    2015-01-01

    Roč. 6, č. 1 (2015), s. 575-587 ISSN 2041-6520 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : tumor suppressor p53 * enzyme amplification reaction * nucleoside triphosphates Subject RIV: CC - Organic Chemistry; BO - Biophysics (BFU-R) Impact factor: 9.144, year: 2015 http://pubs.rsc.org/en/content/articlepdf/2015/sc/c4sc01906g

  9. Croton argenteus preparation inhibits initial growth, mitochondrial respiration and increase the oxidative stress from Senna occidentalis seedlings

    OpenAIRE

    KATLIN S. RECH; CRISTIANE B. SILVA; JULIANA D. KULIK; JOSIANE F.G. DIAS; SANDRA M.W. ZANIN; VITOR A. KERBER; FERNANDA M.M. OCAMPOS; LUCIANE DALARMI; GEDIR O. SANTOS; EUCLÉSIO SIMIONATTO; CRISTINA P. LIMA; OBDÚLIO G. MIGUEL; MARILIS D. MIGUEL

    2015-01-01

    Senna ocidentalis is a weed, native to Brazil, considered to infest crops and plantations, and is responsible for yield losses of several crops, particularly soybean. The aim of this work was to evaluate if theCroton argenteus extract and fractions possess phytotoxic activity on S. ocidentalis. The crude ethanolic extract (CEE) and its hexanic (HF), chloroformic (CLF) and ethyl acetate (EAF) fractions were tested in germination, growth, oxidative stress increase, Adenosine triphosphate, L-mal...

  10. Effectiveness of Devices to Monitor Biofouling and Metals Deposition on Plumbing Materials Exposed to a Full-Scale Drinking Water Distribution System

    OpenAIRE

    Ginige, Maneesha P.; Garbin, Scott; Wylie, Jason; Krishna, K. C. Bal

    2017-01-01

    A Modified Robbins Device (MRD) was installed in a full-scale water distribution system to investigate biofouling and metal depositions on concrete, high-density polyethylene (HDPE) and stainless steel surfaces. Bulk water monitoring and a KIWA monitor (with glass media) were used to offline monitor biofilm development on pipe wall surfaces. Results indicated that adenosine triphosphate (ATP) and metal concentrations on coupons increased with time. However, bacterial diversities decreased. Th...

  11. Free methionine supplementation limits alcohol-induced liver damage in rats

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Bode, C.; Bode, J.C.

    1998-01-01

    .3 to 42.1 +/- 7.2 micromol/g of protein in the alcohol group). Hepatic adenosine triphosphate content increased significantly with higher methionine consumption (13.5 +/- 0.8 vs. 26.9 +/- 2.8 micromol/g of protein in the control group and 11.9 +/- 1.4 vs. 20.5 +/- 2.5 micromol/g of protein in the alcohol...

  12. Single Nutrients and Immunity

    Science.gov (United States)

    1982-02-01

    HA antibody re- sponses. sponse after the administration of Salmonella Rats fed a riboflavin-deficient diet showed pullorum antigens, although no...pools of thymi- rats with severe folic acid deficiency after dine triphosphate and deoxyadenosine tri- inoculation with formalinized R. tiphi in...antigens. with Salmonella typhimurium ( 15 1). Chevalier and Aschkenasy (147) found that Newberne and colleagues (152-155) also a 7% dietary content of

  13. Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

    International Nuclear Information System (INIS)

    Dhar, A.; Paul, A.K.; Shukla, S.D.

    1990-01-01

    Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

  14. Purinergic Signaling Pathways in Endocrine System

    Science.gov (United States)

    Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

    2015-01-01

    Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. PMID:25960051

  15. RNAi mediated gene silencing of ITPA using a targeted nanocarrier: Apoptosis induction in SKBR3 cancer cells.

    Science.gov (United States)

    Charbgoo, Fahimeh; Behmanesh, Mehrdad; Nikkhah, Maryam; Kane, Eric G

    2017-08-01

    A pure nucleotide pool is required for high-fidelity DNA replication and prevention of carcinogenesis in living cells. Human inosine triphosphatase (ITPase), encoded by the ITPA gene, plays a critical role in maintaining the purity of the cellular nucleotide pool by excluding nucleotides that enhance mutagenesis. ITPase is a nucleoside triphosphate pyrophosphatase that hydrolyzes the non-canonical nucleotides inosine triphosphate (ITP) and xanthine triphosphate (XTP). The monophosphate products of ITPase reactions are subsequently excluded from the nucleotide pool and the improper substitution of ITP and XTP into DNA and RNA is prevented. Previous studies show that deficiency in ITPA can suppress cellular growth and enhance DNA instability. In this study, we evaluated the influence of effective ITPA down-regulation on the induction of apoptosis in a human cancer cell line using folate-single wall nanotubes (SWNT) as a targeted nanocarrier. We assessed whether SWNT enhances IPTA-siRNA transfection efficiency in cancer cells using folate as a homing device. Since folate receptor is considerably overexpressed in cancer cells, conjugation of SWNTs to folate could enhance their cancer-specific penetrance. We found that nanocarrier mediated ITPA-siRNA transfection into SKBR3 cells caused significant reduction of ITPA mRNA expression level and complete down-regulation of the ITPase protein product. The silencing of ITPA led to promotion of apoptosis in SWNT-treated SKBR3 cancer cells. © 2017 John Wiley & Sons Australia, Ltd.

  16. Effect of aging on phosphate metabolites of rat brain as revealed by the in vivo and in vitro 31P NMR measurements

    International Nuclear Information System (INIS)

    Liu, Hsiuchih; Chi, Chinwen; Liu, Tsungyun; Liu, Lianghui; Luh, Wenming; Hsieh, Changhuain; Wu, Wenguey

    1991-01-01

    Changes of phosphate metabolism in brains of neonate, weaning and adult rats were compared using both in vivo and in vitro nuclear magnetic resonance spectra. Ratios of phosphocreatine/nucleoside triphosphate (PCr/NTP) were the same in neonatal brain in both in vivo and in vitro studies, but not in weaning and adult brains. This discrepancy may have resulted from extended cerebral hypoxia due to slowed freezing of the brain by the increased skull thickness and brain mass in the weaning and adult rats. Variations of in vitro extraction condition for this age-related study may lead to systematic errors in the adult rats. Nevertheless, the phosphomonoester/nucleoside triphosphate (PME/NTP) ratios in extracts of brain from neonatal rats were higher than those obtained in vivo. In addition, the glycerophosphorylethanolamine plus glycerophosphorylcholine/nucleoside triphosphate (GPE+GPC/NTP) ratios, which were not measurable in vivo, showed age-dependent increase in extracts of rat brain. Some of the phosphomonoester and phosphodiester molecules in rat brain may be undetectable in in vivo NMR analysis because of their interaction with cellular components. The total in vitro GPE and GPC concentration in brain from neonatal rat was estimated to be 0.34 mmole/g wet tissue

  17. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  18. How Native and Alien Metal Cations Bind ATP: Implications for Lithium as a Therapeutic Agent

    Science.gov (United States)

    Dudev, Todor; Grauffel, Cédric; Lim, Carmay

    2017-02-01

    Adenosine triphosphate (ATP), the major energy currency of the cell, exists in solution mostly as ATP-Mg. Recent experiments suggest that Mg2+ interacts with the highly charged ATP triphosphate group and Li+ can co-bind with the native Mg2+ to form ATP-Mg-Li and modulate the neuronal purine receptor response. However, it is unclear how the negatively charged ATP triphosphate group binds Mg2+ and Li+ (i.e. which phosphate group(s) bind Mg2+/Li+) and how the ATP solution conformation depends on the type of metal cation and the metal-binding mode. Here, we reveal the preferred ATP-binding mode of Mg2+/Li+ alone and combined: Mg2+ prefers to bind ATP tridentately to each of the three phosphate groups, but Li+ prefers to bind bidentately to the terminal two phosphates. We show that the solution ATP conformation depends on the cation and its binding site/mode, but it does not change significantly when Li+ binds to Mg2+-loaded ATP. Hence, ATP-Mg-Li, like Mg2+-ATP, can fit in the ATP-binding site of the host enzyme/receptor, activating specific signaling pathways.

  19. Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

    Science.gov (United States)

    Waisertreiger, Irina S.-R.; Menezes, Miriam R.; Randazzo, James; Pavlov, Youri I.

    2010-01-01

    Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA) hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs. PMID:20936128

  20. Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site.

    Science.gov (United States)

    Skurat, A V; Yurkova, M S; Baranova, L A; Gulyaev, N N; Bulargina, T V; Severin, E S

    1985-03-01

    6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.

  1. Purinergic signaling pathways in endocrine system.

    Science.gov (United States)

    Bjelobaba, Ivana; Janjic, Marija M; Stojilkovic, Stanko S

    2015-09-01

    Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. Published by Elsevier B.V.

  2. Characterization of a submitochondrial system of DNA replication

    International Nuclear Information System (INIS)

    Gray, M.S.

    1985-01-01

    A soluble submitochondrial system capable of DNA synthesis was derived from a high salt (1.0M NaCl)-Nonidet-mitoplast lysate and used to study various aspects of mitochondrial DNA synthesis. Incorporation of [ 3 H]dTMP into nacent mitochondrial DNA by the soluble extract requires mitochondrial DNA, the four deoxyribonucleoside triphosphates, magnesium and ATP. The optimal conditions for the incorporation of [ 3 H]dTMP by the soluble extract were, 7.5 μg of mitochrondrial DNA/mg extract protein, 48 μM each of the four deoxyribonucleoside triphosphates, 8 mM magnesium acetate and 3 mM ATP. The maintenance of the ATP level was shown to be essential for the proper replication of mitochondrial DNA and it is postulated that ATP depletion results in reduced DNA ligase activity and/or reduced DNA topoisomerase type 11 activity. Inhibition of mitochondrial DNA synthesis is seen in the presence of 2',3'-dideoxythymidine triphosphate, an inhibitor of DNA polymerase-γ. Aphidicolin, a specific inhibitor of DNA polymerase α, had no inhibitory effect on [ 3 H]dTMP incorporation into mitochondrial DNA. Incorporation of radiolabel is inhibited by pancreatic DNase I, ethidium bromide and novobiocin

  3. DNA repair and replication in cells of Escherichia coli made permeable with hypotonic buffers

    Energy Technology Data Exchange (ETDEWEB)

    Boye, E.

    1980-10-01

    Hypotonic buffers (20 mM phosphate or 40 mM Tris) were found to make cells of Escherichia coli permeable to low-molecular-weight compounds without significantly affecting cell viability. When cells of E. coli were suspended in hypotonic buffers during irradiation no repair of radiation-induced DNA single-strand breaks occurred. Addition of the four deoxyribonucleoside triphosphates and Mg/sup 2/+ after irradiation brings about a recovery of the rejoining ability. NAD stimulated repair in the presence of the four deoxyribonucleoside triphosphates and Mg/sup 2/+, while the presence of ATP had no significant effect. The initial yield of radiation-induced DNA single-strand breaks was increased in cells treated with hypotonic buffers. This is explained by an efflux of a large fraction of the intracellular compounds containing sulfhydryl groups. Cells of E. coli in 40 mM Tris buffer incorporated exogenous deoxyribonucleoside triphosphates at half the rate observed in cells made permeable by toluene. This DNA synthesis occurred in polAl cells and was ATP dependent and thus probably represents semiconservative replication.

  4. Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

    Directory of Open Access Journals (Sweden)

    Irina S.-R. Waisertreiger

    2010-01-01

    Full Text Available Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP deoxynucleoside triphosphate and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs.

  5. Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole.

    Directory of Open Access Journals (Sweden)

    Daniela Scribano

    Full Text Available Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183PAPAP(187 motif of OmpA, nor the N-terminal polyproline (43PPPP(46 motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s. A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.

  6. Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole.

    Science.gov (United States)

    Scribano, Daniela; Petrucca, Andrea; Pompili, Monica; Ambrosi, Cecilia; Bruni, Elena; Zagaglia, Carlo; Prosseda, Gianni; Nencioni, Lucia; Casalino, Mariassunta; Polticelli, Fabio; Nicoletti, Mauro

    2014-01-01

    Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183)PAPAP(187) motif of OmpA, nor the N-terminal polyproline (43)PPPP(46) motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.

  7. The Effects and Pharmacokinetics of Acyclovir in Neonates

    Directory of Open Access Journals (Sweden)

    Gian Maria Pacifici

    2016-12-01

    Full Text Available Acyclovir (9-[2-hydroxyethoxymethyl] guanine is an acyclic nucleoside analogue of guanosine which is a potent and selective antiviral agent. Acycloviris converted to the monophosphate by thymidine kinase the virus-specific form of this enzyme and is subsequently converted to the triphosphate by the host cell kinase. Acyclovir triphosphate inhibits viral DNA-polymerase terminating the chain and is the active form. It is 30 times more potent against the herpes simplex virus enzyme than the host enzyme. Acyclovir triphosphate is fairly rapidly broken down within the host cells by cellular phosphatases. Resistance due to changes in the viral genes coding for thymidine kinase or DNA polymerase cause acyclovir-resistant herpes simplex virus and has been the cause of pneumonia, encephalitis and mucocutaneous infections. Acyclovir can be administered orally or intravenously. When it is given orally, only 10-20% of the dose is absorbed. Acyclovir is widely distributed throughout the body, reaching concentrations in the cerebrospinal fluid which are 30 to 50% of those in the serum. In neonates, the half-life of acyclovir is about 5 hours, but it is 2.5 hours in children over 3 months old. The herpes simplex virus is transmitted vertically from infected mothers to fetuses and the administration of 400 mg acyclovir orally three times daily from 36 weeks of pregnancy until delivery has been suggested. Alternatively, a cesarean section can be performed to avoid the transmission of the herpes simplex virus to fetuses. The aim of this study is to review the effects and pharmacokinetics of acyclovir in neonates.

  8. Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Weiler, Monica [Department of Medicine III and Transfusion Medicine, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich (Germany); Schmetzer, Helga [Helmholtz Center Munich (Germany); German Research Center for Environmental Health, Munich (Germany); Braeu, Marion; Buhmann, Raymund [Helmholtz Center Munich (Germany); German Research Center for Environmental Health, Munich (Germany); Department of Medicine III and Transfusion Medicine, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich (Germany)

    2016-11-15

    The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

  9. Timescale of silver nanoparticle transformation in neural cell cultures impacts measured cell response

    Energy Technology Data Exchange (ETDEWEB)

    Hume, Stephanie L.; Chiaramonti, Ann N.; Rice, Katherine P.; Schwindt, Rani K. [National Institute of Standards and Technology (NIST), Applied Chemicals and Materials Division (United States); MacCuspie, Robert I. [National Institute of Standards and Technology (NIST), Materials Measurement Science Division (United States); Jeerage, Kavita M., E-mail: jeerage@boulder.nist.gov [National Institute of Standards and Technology (NIST), Applied Chemicals and Materials Division (United States)

    2015-07-15

    Both serum protein concentration and ionic strength are important factors in nanoparticle transformation within cell culture environments. However, silver nanoparticles are not routinely tracked at their working concentration in the specific medium used for in vitro toxicology studies. Here we evaluated the transformation of electrostatically stabilized citrate nanoparticles (C-AgNPs) and sterically stabilized polyvinylpyrrolidone nanoparticles (PVP-AgNPs) in a low-serum (∼ 0.2 mg/mL bovine serum albumin) culture medium, while measuring the response of rat cortex neural progenitor cells, which differentiate in this culture environment. After 24 h, silver nanoparticles at concentrations up to 10 µg/mL did not affect adenosine triphosphate levels, whereas silver ions decreased adenosine triphosphate levels at concentrations of 1.1 µg/mL or higher. After 240 h, both silver nanoparticles, as well as silver ion, unambiguously decreased adenosine triphosphate levels at concentrations of 1 and 1.1 µg/mL, respectively, suggesting particle dissolution. Particle transformation was investigated in 1:10 diluted, 1:2 diluted, or undiluted differentiation medium, all having an identical protein concentration, to separate the effect of serum protein stabilization from ionic strength destabilization. Transmission electron microscopy images indicated that particles in 1:10 medium were not surrounded by proteins, whereas particles became clustered within a non-crystalline protein matrix after 24 h in 1:2 medium and at 0 h in undiluted medium. Despite evidence for a protein corona, particles were rapidly destabilized by high ionic strength media. Polyvinylpyrrolidone increased the stability of singly dispersed particles compared to citrate ligands; however, differences were negligible after 4 h in 1:2 medium or after 1 h in undiluted medium. Thus low-serum culture environments do not provide sufficient colloidal stability for long-term toxicology studies with citrate

  10. The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

    Science.gov (United States)

    Rozovskaya, T A; Rechinsky, V O; Bibilashvili, R S; Karpeisky MYa; Tarusova, N B; Khomutov, R M; Dixon, H B

    1984-01-01

    DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted. Images Fig. 1. Fig. 2. PMID:6083781

  11. Timescale of silver nanoparticle transformation in neural cell cultures impacts measured cell response

    International Nuclear Information System (INIS)

    Hume, Stephanie L.; Chiaramonti, Ann N.; Rice, Katherine P.; Schwindt, Rani K.; MacCuspie, Robert I.; Jeerage, Kavita M.

    2015-01-01

    Both serum protein concentration and ionic strength are important factors in nanoparticle transformation within cell culture environments. However, silver nanoparticles are not routinely tracked at their working concentration in the specific medium used for in vitro toxicology studies. Here we evaluated the transformation of electrostatically stabilized citrate nanoparticles (C-AgNPs) and sterically stabilized polyvinylpyrrolidone nanoparticles (PVP-AgNPs) in a low-serum (∼ 0.2 mg/mL bovine serum albumin) culture medium, while measuring the response of rat cortex neural progenitor cells, which differentiate in this culture environment. After 24 h, silver nanoparticles at concentrations up to 10 µg/mL did not affect adenosine triphosphate levels, whereas silver ions decreased adenosine triphosphate levels at concentrations of 1.1 µg/mL or higher. After 240 h, both silver nanoparticles, as well as silver ion, unambiguously decreased adenosine triphosphate levels at concentrations of 1 and 1.1 µg/mL, respectively, suggesting particle dissolution. Particle transformation was investigated in 1:10 diluted, 1:2 diluted, or undiluted differentiation medium, all having an identical protein concentration, to separate the effect of serum protein stabilization from ionic strength destabilization. Transmission electron microscopy images indicated that particles in 1:10 medium were not surrounded by proteins, whereas particles became clustered within a non-crystalline protein matrix after 24 h in 1:2 medium and at 0 h in undiluted medium. Despite evidence for a protein corona, particles were rapidly destabilized by high ionic strength media. Polyvinylpyrrolidone increased the stability of singly dispersed particles compared to citrate ligands; however, differences were negligible after 4 h in 1:2 medium or after 1 h in undiluted medium. Thus low-serum culture environments do not provide sufficient colloidal stability for long-term toxicology studies with citrate

  12. Depression of presynaptic excitation by the activation of vanilloid receptor 1 in the rat spinal dorsal horn revealed by optical imaging

    Directory of Open Access Journals (Sweden)

    Ikeda Hiroshi

    2006-02-01

    Full Text Available Abstract In this study, we show that capsaicin (CAP depresses primary afferent fiber terminal excitability by acting on vanilloid receptor 1 (TRPV1 channels of primary afferent fibers in adenosine 5'-triphosphate (ATP- and temperature-dependent manner using two optical imaging methods. First, transverse slices of spinal cord were stained with a voltage-sensitive dye and the net excitation in the spinal dorsal horn was recorded. Prolonged treatment (>20 min with the TRPV1 channel agonist, CAP, resulted in a long-lasting inhibition of the net excitation evoked by single-pulse stimulation of C fiber-activating strength. A shorter application of CAP inhibited the excitation in a concentration-dependent manner and the inhibition was reversed within several minutes. This inhibition was Ca++-dependent, was antagonized by the TRPV1 channel antagonist, capsazepine (CPZ, and the P2X and P2Y antagonist, suramin, and was facilitated by the P2Y agonist, uridine 5'-triphosphate (UTP. The inhibition of excitation was unaffected by bicuculline and strychnine, antagonists of GABAA and glycine receptors, respectively. Raising the perfusate temperature to 39°C from 27°C inhibited the excitation (-3%/°C. This depressant effect was antagonized by CPZ and suramin, but not by the P2X antagonist, 2', 3'-O-(2,4,6-trinitrophenyl adenosine 5'-triphosphate (TNP-ATP. Second, in order to record the presynaptic excitation exclusively, we stained the primary afferent fibers anterogradely from the dorsal root. CAP application and a temperature increase from 27°C to 33°C depressed the presynaptic excitation, and CPZ antagonized these effects. Thus, this study showed that presynaptic excitability is modulated by CAP, temperature, and ATP under physiological conditions, and explains the reported central actions of CAP. These results may have clinical importance, especially for the control of pain.

  13. Energetic and cell membrane metabolic products in patients with primary insomnia: a 31-phosphorus magnetic resonance spectroscopy study at 4 tesla.

    Science.gov (United States)

    Harper, David G; Plante, David T; Jensen, J Eric; Ravichandran, Caitlin; Buxton, Orfeu M; Benson, Kathleen L; O'Connor, Shawn P; Renshaw, Perry F; Winkelman, John W

    2013-04-01

    Primary insomnia (PI) is a sleep disorder characterized by difficulty with sleep initiation, maintenance, and/or the experience of nonrestorative sleep combined with a subsequent impairment of daytime functioning. The hyperarousal hypothesis has emerged as the leading candidate to explain insomnia symptoms in the absence of specific mental, physical, or substance-related causes. We hypothesized that the cellular energetic metabolites, including beta nucleoside triphosphate, which in magnetic resonance spectroscopy approximates adenosine triphosphate (ATP), and phosphocreatine (PCr), would show changes in PI reflecting increased energy demand. Matched-groups, cross-sectional study performed at two university-based hospitals. Sixteen medication-free individuals (eight males, eight females; mean ± standard deviation (SD) age = 37.2 ± 8.4 y) with PI and 16 good sleepers (nine males, seven females; mean ± SD age = 37.6 ± 4.7 y). Diagnosis was established for all individuals by unstructured clinical interview, Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (SCID), sleep diary, and actigraphy. Polysomnography was collected in individuals with PI. Phosphorous magnetic resonance spectroscopy (31P MRS) data were collected on all individuals at 4 Tesla. We assessed cell membrane (anabolic precursors and catabolic metabolites) and bioenergetic (ATP, phosphocreatine) metabolites in gray matter and white matter to determine their relationship to the presence and severity of PI. Individuals with PI showed lower phosphocreatine in gray matter and an unexpected decrease of phosphocholine, a precursor of the cell membrane compound phosphatidylcholine, in white matter. In addition, there was a trend toward a negative association between polysomnographically determined wake after sleep onset and gray matter beta-nucleoside triphosphate and white matter phosphocholine in the primary insomnia group. These results support the

  14. Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adnosine 5-diphosphate (ADP) in human platelets.

    Science.gov (United States)

    Park, H S; Hourani, S M

    1999-07-01

    Adenosine 5'-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were measured and compared in washed platelets using an aggregometer. ADP and its analogues 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP), adenosine 5'(alpha,beta-methylene)diphosphonate (AMPCP), S(P)-adenosine 5'-O-(1-thiodiphosphate) (AD-P alphaS) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) were used as agonists. Adenosine 5'-triphosphate (ATP) and its analogues, P1, P5-diadenosine pentaphosphate (ApsA), adenosine (5'-(alpha,beta-methylene)triphosphonate (AMPCPP), 2-methylthioadenosine 5'-triphosphate (2-methylthio-ATP) and uridine 5'-triphosphate (UTP), as well as the trypanocidal drug suramin, were used as antagonists. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. ADPalphaS and ADPbetaS were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPalphaS antagonized ADP-induced aggregation with an apparent pK(B) value of 5.5+/-0.1. 2-Methylthio-ATP also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change with an apparent pK(B) value of 7.0+/-0.2 for aggregation and 5.2+/-0.2 for shape change. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y1 receptor) with aggregation requiring the activation of two receptors (the P2Y1 and another P2Y receptor).

  15. Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets.

    Science.gov (United States)

    Bischler, Thorsten; Hsieh, Ping-Kun; Resch, Marcus; Liu, Quansheng; Tan, Hock Siew; Foley, Patricia L; Hartleib, Anika; Sharma, Cynthia M; Belasco, Joel G

    2017-02-03

    RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

    International Nuclear Information System (INIS)

    Weiler, Monica; Schmetzer, Helga; Braeu, Marion; Buhmann, Raymund

    2016-01-01

    The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3 + T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP and GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.

  17. Assembly of IMPDH2-based, CTPS-based, and mixed rod/ring structures is dependent on cell type and conditions of induction.

    Science.gov (United States)

    Keppeke, Gerson Dierley; Calise, S John; Chan, Edward K L; Andrade, Luis Eduardo C

    2015-06-20

    Inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) biosynthetic pathways induces cells to assemble rod/ring (RR) structures, also named cytoophidia, which consist of the enzymes cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). We aim to explore the interaction of CTPS and IMPDH2 in the generation of RR structures. HeLa and COS-7 cells were cultured in normal conditions or in the presence of 6-diazo-5-oxo-L-norleucine (DON), ribavirin, or mycophenolic acid (MPA). Over 90% of DON-treated cells presented RR structures. In HeLa cells, 35% of the RR structures were positive for IMPDH2 alone, 26% were CTPS alone, and 31% were IMPDH2/CTPS mixed, while in COS-7 cells, 42% of RR were IMPDH2 alone, 41% were CTPS alone, and 10% were IMPDH2/CTPS mixed. Ribavirin and MPA treatments induced only IMPDH2-based RR. Cells were also transfected with an N-terminal hemagglutinin (NHA)-tagged CTPS1 construct. Over 95% of NHA-CTPS1 transfected cells with DON treatment presented IMPDH2-based RR and almost 100% presented CTPS1-based RR; when treated with ribavirin, over 94% of transfected cells presented IMPDH2-based RR and 37% presented CTPS1-based RR, whereas 2% of untreated transfected cells presented IMPDH2-based RR and 28% presented CTPS1-based RR. These results may help in understanding the relationship between CTP and GTP biosynthetic pathways, especially concerning the formation of filamentous RR structures. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  18. Improving the Diet for the Rearing of Glossina brevipalpis Newstead and Glossina austeni Newstead: Blood Source and Collection – Processing – Feeding Procedures

    Science.gov (United States)

    De Beer, Chantel J.; Venter, Gert J.; Vreysen, Marc J. B.

    2016-01-01

    One of the challenges to maintain tsetse fly (Diptera: Glossinidae) colonies is the sustainable supply of high quality blood meals. The effect of using anticoagulants during collection of the blood, the addition of phagostimulants to the blood meals as well as using mixtures of bovine and porcine blood in different proportions for feeding on colony productivity was assessed. Defibrinated bovine blood was found to be suitable to maintain both the Glossina brevipalpis Newstead and Glossina austeni Newstead colonies. Blood collected with the anticoagulants sodium citrate, citric sodium combination, citrate phosphate dextrose adenine and citric acid did not affect colony performance of both species. Defibrinated bovine and porcine blood in a 1:1 ratio or the feeding of either bovine or porcine blood on alternating days improved pupae production of G. austeni and can be used to enhance colony growth. Bovine blood is appropriate to maintain G. brevipalpis colonies, however, feeding either bovine or porcine blood on alternating days did improve productivity. Adding the phagostimulants inosine tri-phosphate, cytosine mono-phosphate and guanosine mono-phosphate to the blood at a concentration of 10−4 M improved pupae production of the G. brevipalpis colony. The addition of adenosine tri-phosphate and inosine tri-phosphate improved the performance of the G. austeni colony. Decisions on the most suitable rearing diet and feeding protocols will not only depend on the biological requirements of the species but also on the continuous supply of a suitable blood source that can be collected and processed in a cost-effective way. PMID:28006007

  19. The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Javid-Majd, Farah; Yang, Dong [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States); Ioerger, Thomas R. [Department of Computer Science, Texas A& M University, College Station, Texas 77843-2128 (United States); Sacchettini, James C., E-mail: sacchett@tamu.edu [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States)

    2008-06-01

    The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

  20. Retrograde hot-shot cardioplegia in patients with left ventricular hypertrophy undergoing aortic valve replacement.

    Science.gov (United States)

    Ascione, Raimondo; Suleiman, Saadeh M; Angelini, Gianni D

    2008-02-01

    Intermittent antegrade cold-blood cardioplegia followed by terminal warm-blood cardioplegic reperfusion or hot-shot is reported to reduce myocardial injury in the setting of coronary surgery. The efficacy of this cardioplegic technique in patients with left ventricular hypertrophy secondary to aortic stenosis remains uncertain. Thirty-six patients with left ventricular hypertrophy undergoing aortic valve replacement were prospectively randomized to cold-blood cardioplegia either alone (cold-blood cardioplegia group) or with retrograde hot-shot (hot-shot group). Reperfusion injury was assessed by measuring myocardial levels of adenosine triphosphate and lactate in left and right ventricular biopsies taken 5 minutes after institution of cardiopulmonary bypass and 20 minutes after removal of cross-clamp using high-performance liquid chromatography and enzymatic techniques. Myocardial injury was assessed by serial release of troponin I up to 48 hours postoperatively. Overall clinical outcome was prospectively collected. Baseline and intraoperative characteristics were similar between groups. In the hot-shot group, there were no significant changes in the myocardial concentration of adenosine triphosphate and lactate in both left and right ventricular biopsies after reperfusion. In the cold-blood cardioplegia group, there was a trend to a fall in adenosine triphosphate levels in the left and right ventricular biopsies after reperfusion, but this reached statistical significance only in the right ventricle. Troponin I release was raised in both groups at 4 and 12 hours after surgery (p < 0.05), but did not reach levels of myocardial infarction. The terminal retrograde hot-shot reperfusion does not add any extra benefit to antegrade cold-blood cardioplegia in preventing myocardial injury in patients with left ventricular hypertrophy undergoing aortic valve replacement. Nevertheless, it appears to reduce ischemic stress in the right ventricle. There was no difference in

  1. Kinetics of the stages of transcription initiation at the Escherichia coli lac UV5 promoter

    International Nuclear Information System (INIS)

    Straney, S.B.; Crothers, D.M.

    1987-01-01

    The kinetics of initiation by Escherichia coli RNA polymerase on the lac L8UV5 promoter was studied by a gel retardation method that separates protein-DNA complexes from free DNA. The binding constant of the closed complex, the forward and reverse rate constants of isomerization from closed to open complex, and the forward rate constant from the open to initiated complex were measured. Both the forward and reverse isomerization rates were found to be temperature dependent, and the activation energies for these steps were determined. The rates of open complex formation and dissociation were not affected by the addition of ribonucleotide triphosphates; however, the extent of dissociation was greatly reduced if the triphosphates added allowed a short, unstable RNA product to form. The dissociation rate was not affected by heparin, a polyanion competitor that sequesters the polymerase. The rate of initiated complex formation appeared to be dependent on whether the initiating moiety was a mononucleotide triphosphate or dinucleoside monophosphate and on the sequence of the dinucleoside. These results are compared to those found on both the lac L8UV5 and other bacterial and phage promoters by less direct measurements. We use the values obtained for the individual rate constants to investigate the predicted steady-state kinetics of initiation-limited transcription, with the conclusion that the rate-limiting step is formation of the open complex in the limit of low polymerase concentration. However, when RNA polymerase is saturating, the rate is determined by the transition from open complex into the stably initiated ternary complex

  2. Labelled radioactive adenosinphosphates for the determination of toxic action

    International Nuclear Information System (INIS)

    Tahbaz, Z.

    1983-01-01

    Normal house-flies had been fed with carrier free radiophosphate (phosphorus). Many phosphorous containing substances in the tissue of the housefly are labelled with radiophosphorus by this procedure. Radiophosphorus is also found in the nucleotides of the housefly after applying radioactive phosphate. Suitable methods for processing and separation had been selected and worked out to isolate 32 P-adenosin-triphosphate 32 P-adenosin-diphosphate, 32 P-adenosin-monophosphate and 32 P-phosphate. Working at low temperature prevents chemical changes of the nucleotides. Extraction and thin layer chromatorgraphy turned out to be effective separation procedures for preparing samples for radioactivity measurement of the nucleotides. Autoradiographic techniques, scanning and liquid scitillation counting had been used for radioactivity measurements of the radioactive zones at the chromatograms. The results of these measurements provide information concerning the normal composition of adenosin-phosphates in the tissues of the housefly. If the animals are exposed to toxic chemicals, to insecticides, the composition of the phosphate containing compounds is changing. The concentration of adenosin-triphosphate is decreasing and the concentration of phosphate is increasing. This can be very easily shown by scanning the chromatograms of the extracts of the muscles of houseflies after feeding the animals with radioactive phosphate. Using this method, it is possible to show the toxic action of insecticides upon the metabolism of adenosin-phosphates. The decrease of the radioactivity at the zone of the adenosin-triphosphate and the increase of the radioactivity at the phosphate zone corresponds to the toxic action of foreign chemicals like insecticides. By using this tracer technique, it may be possible to investigate the toxic action of several toxic chemicals, if they are applied at the same time, thus investigating synergetic actions of environmental poisons. (Author)

  3. Immune Responses and Protection of Aotus Monkeys Immunized with Irradiated Plasmodium vivax Sporozoites

    Science.gov (United States)

    2011-01-01

    concentration of 1 J.lg/mL in PBS. Plates were then blocked with 5% skim milk in PBS, pH 7.4, for 2 hrs at room tempera- ture. After the plates were washed...J.!L using a mix of 2 mM MgCl2/50 mM KC1/10 mM Tris pH 8.3 (HC1 )/125 J.!M of each of the four deoxyribonucleotide triphosphates/250 nM of each...Liliana Soto, Fabian Mendez, Myriam Arevalo-Herrera, and Socrates Herrera, lnstituto de lnmunologia. Edificio de Microbiologia, Facultad de Salud

  4. GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein

    DEFF Research Database (Denmark)

    Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara

    2011-01-01

    transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin a/ß pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O......-(thio)triphosphate (GTP¿S). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which...

  5. Pathophysiological effects of RhoA and Rho-associated kinase on cardiovascular system.

    Science.gov (United States)

    Cai, Anping; Li, Liwen; Zhou, Yingling

    2016-01-01

    In past decades, growing evidence from basic and clinical researches reveal that small guanosine triphosphate binding protein ras homolog gene family, member A (RhoA) and its main effector Rho-associated kinase (ROCK) play central and complex roles in cardiovascular systems, and increasing RhoA and ROCK activity is associated with a broad range of cardiovascular diseases such as congestive heart failure, atherosclerosis, and hypertension. Favorable outcomes have been observed with ROCK inhibitors treatment. In this review, we briefly summarize the pathophysiological roles of RhoA/ROCK signaling pathway on cardiovascular system, displaying the potential benefits in the cardiovascular system with controlling RhoA/ROCK signaling pathway.

  6. Dependence of anaphylactic histamine release from rat mast cells on cellular energy metabolism

    DEFF Research Database (Denmark)

    Johansen, Torben

    1981-01-01

    The relation between anaphylactic histamine release and the adenosine triphosphate (ATP) content of the mast cells was studied. The cells were incubated with glycolytic (2-deoxyglucose) and respiratory inhibitors (antimycin A and oligomycin) in order to decrease the ATP content of the cells prior...... pretreated with 2-deoxyglucose. The release of histamine from these cells was reduced when respiratory inhibitors were added to the cell suspension 5 to 20 sec after exposure of the cells to antigen. This may indicate that the secretory process requires energy, and it seems necessary that energy should...... be produced as the release of histamine takes place....

  7. Inhibition of cell proliferation through an ATP-responsive co-delivery system of doxorubicin and Bcl-2 siRNA

    OpenAIRE

    Zhang, Jianxu; Wang, Yudi; Chen, Jiawen; Liang, Xiao; Han, Haobo; Yang, Yan; Li, Quanshun; Wang, Yanbo

    2017-01-01

    Jianxu Zhang,1,2,* Yudi Wang,2,* Jiawen Chen,2 Xiao Liang,2 Haobo Han,1,2 Yan Yang,2 Quanshun Li,1,2 Yanbo Wang1 1Department of Urology, First Hospital of Jilin University, 2Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun, People’s Republic of China *These authors contributed equally to the work Abstract: Herein, DNA duplex was constructed through the hybridization of adenosine triphosphate (AT...

  8. Biological assessment of radiation damage of ATP by soft X-rays

    International Nuclear Information System (INIS)

    Akimitsu, Nobuyoshi; Fujii, Shin-ichiro; Fujii, Kentaro; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2011-01-01

    ATP (Adenosine tri-phosphate), one of ribonucleic acids, acts as an intracellular energy transfer. ATP is used as a substrate to synthesized messenger RNA and as a ligand of inter-cellular signaling. SPring-8 beam line BL23SU can produce high resolution monochromatic soft X-rays. In this study, we analyzed the soft X-ray induced radiation damage of ATP by various biological assessments, such as energy donor activity, genetic information transfer activity and inter-cellular signaling activity. (author)

  9. K-ATP channel expression and pharmacological in vivo and in vitro studies of the K-ATP channel blocker PNU-37883A in rat middle meningeal arteries

    DEFF Research Database (Denmark)

    Ploug, K.B.; Boni, L.J.; Baun, M.

    2008-01-01

    Background and purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K+ (K-ATP) channel opening may cause migraine by dilatating...... intracranial arteries, including the middle meningeal artery (MMA). We studied the K-ATP channel expression profile in rat MMA and examined the potential inhibitory effects of the K-ATP channel blocker PNU-37883A on K-ATP channel opener-induced relaxation of the rat MMA, using the three K-ATP channel openers...

  10. Detection of microbial contaminations in drinking water using ATP measurements – evaluating potential for online monitoring

    DEFF Research Database (Denmark)

    Vang, Óluva Karin; Corfitzen, Charlotte B.; Albrechtsen, Hans-Jørgen

    2011-01-01

    of reagent kit. Surface water diluted 100-1000 times was detected in drinking water with ATP measurements. ATP has the potential as an early warning tool, especially in the period when the contamination concentration is high. 2011 © American Water Works Association AWWA WQTC Conference Proceedings All Rights......There is an increasing call for fast and reliable methods for continuous monitoring of microbial drinking water quality in order to protect public health. The potential for Adenosine triphosphate (ATP) measurements as a real-time analysis for continuous monitoring of microbial drinking water...

  11. P2X receptors

    OpenAIRE

    North, R. Alan

    2016-01-01

    Extracellular adenosine 5′-triphosphate (ATP) activates cell surface P2X and P2Y receptors. P2X receptors are membrane ion channels preferably permeable to sodium, potassium and calcium that open within milliseconds of the binding of ATP. In molecular architecture, they form a unique structural family. The receptor is a trimer, the binding of ATP between subunits causes them to flex together within the ectodomain and separate in the membrane-spanning region so as to open a central channel. P2...

  12. Recombinant human erythropoietin increases survival and reduces neuronal apoptosis in a murine model of cerebral malaria

    DEFF Research Database (Denmark)

    Wiese, Lothar; Hempel, Casper; Penkowa, Milena

    2008-01-01

    BACKGROUND: Cerebral malaria (CM) is an acute encephalopathy with increased pro-inflammatory cytokines, sequestration of parasitized erythrocytes and localized ischaemia. In children CM induces cognitive impairment in about 10% of the survivors. Erythropoietin (Epo) has - besides of its well known...... with recombinant human Epo (rhEpo; 50-5000 U/kg/OD, i.p.) at different time points. The effect on survival was measured. Brain pathology was investigated by TUNEL (Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labelling), as a marker of apoptosis. Gene...

  13. Human DNA polymerase alpha uses a combination of positive and negative selectivity to polymerize purine dNTPs with high fidelity

    Czech Academy of Sciences Publication Activity Database

    Beckman, J.; Kincaid, K.; Hocek, Michal; Spratt, T.; Engels, J.; Cosstick, R.; Kuchta, R. D.

    2007-01-01

    Roč. 46, č. 2 (2007), s. 448-460 ISSN 0006-2960 R&D Projects: GA MŠk LC512 Grant - others:NIH(US) GM54194; NIH(US) TW007372-01; Army Research Office(US) W911NF-05-1-0172; DFG(DE) SFB 579 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA polymerase * nucleoside triphosphates * modified nucleobases Subject RIV: CC - Organic Chemistry Impact factor: 3.368, year: 2007

  14. Upregulation of Human Endogenous Retrovirus-K Is Linked to Immunity and Inflammation in Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Saito, Toshie; Miyagawa, Kazuya; Chen, Shih-Yu; Tamosiuniene, Rasa; Wang, Lingli; Sharpe, Orr; Samayoa, Erik; Harada, Daisuke; Moonen, Jan-Renier A J; Cao, Aiqin; Chen, Pin-I; Hennigs, Jan K; Gu, Mingxia; Li, Caiyun G; Leib, Ryan D; Li, Dan; Adams, Christopher M; Del Rosario, Patricia A; Bill, Matthew; Haddad, Francois; Montoya, Jose G; Robinson, William H; Fantl, Wendy J; Nolan, Garry P; Zamanian, Roham T; Nicolls, Mark R; Chiu, Charles Y; Ariza, Maria E; Rabinovitch, Marlene

    2017-11-14

    Immune dysregulation has been linked to occlusive vascular remodeling in pulmonary arterial hypertension (PAH) that is hereditary, idiopathic, or associated with other conditions. Circulating autoantibodies, lung perivascular lymphoid tissue, and elevated cytokines have been related to PAH pathogenesis but without a clear understanding of how these abnormalities are initiated, perpetuated, and connected in the progression of disease. We therefore set out to identify specific target antigens in PAH lung immune complexes as a starting point toward resolving these issues to better inform future application of immunomodulatory therapies. Lung immune complexes were isolated and PAH target antigens were identified by liquid chromatography tandem mass spectrometry, confirmed by enzyme-linked immunosorbent assay, and localized by confocal microscopy. One PAH antigen linked to immunity and inflammation was pursued and a link to PAH pathophysiology was investigated by next-generation sequencing, functional studies in cultured monocytes and endothelial cells, and hemodynamic and lung studies in a rat. SAM domain and HD domain-containing protein 1 (SAMHD1), an innate immune factor that suppresses HIV replication, was identified and confirmed as highly expressed in immune complexes from 16 hereditary and idiopathic PAH versus 12 control lungs. Elevated SAMHD1 was localized to endothelial cells, perivascular dendritic cells, and macrophages, and SAMHD1 antibodies were prevalent in tertiary lymphoid tissue. An unbiased screen using metagenomic sequencing related SAMHD1 to increased expression of human endogenous retrovirus K (HERV-K) in PAH versus control lungs (n=4). HERV-K envelope and deoxyuridine triphosphate nucleotidohydrolase mRNAs were elevated in PAH versus control lungs (n=10), and proteins were localized to macrophages. HERV-K deoxyuridine triphosphate nucleotidohydrolase induced SAMHD1 and proinflammatory cytokines (eg, interleukin 6, interleukin 1β, and tumor

  15. Analysis of the presence of organic matter (ATP in mobile devices of healthcare workers in hospitals

    Directory of Open Access Journals (Sweden)

    Eunice Beatriz Martin Chaves

    2017-04-01

    Full Text Available We report the result of an awareness campaign about the importance of hygiene of hands after using different devices used in assistance to patients, such as tablets, notebooks, mobile phones, identification, watches, cameras, showing the level of contamination found after technical using detection of ATP (Adenosine triphosphate via bioluminescence, which enables a quantitative assessment ready and easy to implement. ATP molecules found in all living cells react with the enzyme complex generating light representing the presence of organic matter in such objects. Keywords: Nosocomial infection; fomites; hand hygiene

  16. P-31 MR spectroscopy of alcoholic liver diseases

    International Nuclear Information System (INIS)

    Meyerhoff, D.J.; Boska, M.D.; Thomas, A.; Twieg, D.B.; Rockey, D.; Weiner, M.W.

    1988-01-01

    The goal of the experiments was to measure absolute molar metabolite concentrations of phosphorus metabolites in livers of healthy humans and patients with acute alcoholic hepatitis and hepatitis cirrhosis. Quantitative P-31 MR spectroscopy was performed at 2.0 T with a surface coil and the ISIS technique. Absolute hepatic P-31 metabolite concentrations in patients with alcoholic hepatitis and cirrhosis were decreased by 20% - 40% compared with levels in controls. Therefore, these experiments demonstrate that alcoholic liver disease is associated with decreased concentrations of adenosine triphosphate and other phosphorylated metabolites

  17. Amplification and Re-Generation of LNA-Modified Libraries

    DEFF Research Database (Denmark)

    Doessing, Holger; Hansen, Lykke H.; Veedu, Rakesh N.

    2012-01-01

    Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could...... be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of ‘mock’ in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We...

  18. The dynamics of acid-soluble phosphorus compounds in the course of winter and spring wheat germination under various thermic conditions. Part II. Labile phosphorus after hydrolysis of the acid-soluble fraction

    Directory of Open Access Journals (Sweden)

    A. Barbaro

    2015-06-01

    Full Text Available The changes in labile phosphorus compounds content during germination of wheat were investigated. These compounds were determined in acid-soluble germ extracts separated into fractions according to the solubility of their barium salts. Low germination temperature was found to raise the labile phosphorus content in the fraction of insoluble barium salts. If we assume that labile P of this fraction consisted mainly of adenosinedi- and triphosphates, it would seem that the rise, in the ATP and ADP level under the influence of low temperature may be essential for initiating flowering in winter varieties.

  19. Purinergic Signaling Pathways in Endocrine System

    OpenAIRE

    Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

    2015-01-01

    Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors,...

  20. Autoradiography of P2x ATP receptors in the rat brain.

    OpenAIRE

    Balcar, V. J.; Li, Y.; Killinger, S.; Bennett, M. R.

    1995-01-01

    1. Binding of a P2x receptor specific radioligand, [3H]-alpha,beta-methylene adenosine triphosphate ([3H]-alpha,beta-MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non-specific binding was very low (< 7% at 10 nM ligand concentration). 2. The binding was completely inhibited by suramin (IC50 approximately 14-26 microM) but none of the ligands specific for P2y receptors such as 2-methylthio-adenosi...

  1. Investigating structural changes induced by nucleotide binding to RecA using difference FTIR.

    OpenAIRE

    Butler, Blaine C; Hanchett, Ross H; Rafailov, Helena; MacDonald, Gina

    2002-01-01

    Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5'-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5'-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide b...

  2. Platelet Dysfunction in Patients with Chronic Myeloid Leukemia: Does Imatinib Mesylate Improve It?

    OpenAIRE

    Olga Meltem Akay; Fezan Mutlu; Zafer Gülbaş

    2016-01-01

    Objective: The aim of this study was to investigate the effects of imatinib mesylate on platelet aggregation and adenosine triphosphate (ATP) release in chronic myeloid leukemia patients. Materials and Methods: Platelet aggregation and ATP release induced by 5.0 mM adenosine diphosphate, 0.5 mM arachidonic acid, 1.0 mg/ mL ristocetin, and 2 µg/mL collagen were studied by whole blood platelet lumi-aggregometer in 20 newly diagnosed chronic myeloid leukemia patients before ...

  3. TNP-AMP Binding to the Sarcoplasmic Reticulum Ca2+-ATPase Studied by Infrared Spectroscopy

    OpenAIRE

    Liu, Man; Barth, Andreas

    2003-01-01

    Infrared spectroscopy was used to monitor the conformational change of 2′,3′-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca2+-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by β,γ-iminoadenosine 5′-triphosphate (AMPPNP) after AMPPNP release from P3-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of...

  4. Characterization of the effects of adenosine 5'-[beta-thio]-diphosphate in rat liver.

    OpenAIRE

    Keppens, S.; Vandekerckhove, A.; De Wulf, H.

    1993-01-01

    1. In rat liver cells micromolar concentrations of adenosine 5'-[beta-thio]diphosphate (ADP beta S), activate glycogen phosphorylase by an adenosine 3':5'-cyclic monophosphate (cyclic AMP)- independent mechanism. 2. As with adenosine 5'-triphosphate (ATP), ADP beta S also inhibits the rise in cyclic AMP after glucagon. 3. Cytosolic Ca2+ measured in single cells is rapidly increased with a pattern similar for ADP beta S and for ATP. 4. At variance with ATP, ADP beta S hardly increases inositol...

  5. Treadmilling of actin filaments via Brownian dynamics simulations

    DEFF Research Database (Denmark)

    Guo, Kunkun; Shillcock, Julian C.; Lipowsky, Reinhard

    2010-01-01

    Actin polymerization is coupled to the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (Pi). Therefore, each protomer within an actin filament can attain three different nucleotide states corresponding to bound ATP, ADP / Pi, and ADP....... These protomer states form spatial patterns on the growing (or shrinking) filaments. Using Brownian dynamics simulations, the growth behavior of long filaments is studied, together with the associated protomer patterns, as a function of ATP-actin monomer concentration, CT, within the surrounding solution...

  6. The effect of dipolar interaction on the magnetic isotope effect

    DEFF Research Database (Denmark)

    Mojaza, Matin; Pedersen, Jørgen Boiden; Lukzen, Nikita

    2010-01-01

    A multi-channel kinetic description is used to study the magnetic isotope effect (MIE) in zero magnetic field. The maximal isotope effect is equal to the number of channels, two for the hyperfine interaction but four for the electron spin dipole–dipole interaction of the intermediate radical pair....... Quantum mechanical calculations agree with these conclusion and show that large MIE may be obtained even in the presence of a strong exchange interaction. The observed magnesium isotope effect on the rate of enzymatic synthesis of adenosine triphosphate (ATP) is approximately 3 implying that the dipolar...... interaction is responsible for the effect. Our calculations provide support for the proposed mechanism....

  7. Sequencing of Isotope-Labeled Small RNA Using Femtosecond Laser Ablation Time-of-Flight Mass Spectrometry

    Science.gov (United States)

    Kurata-Nishimura, Mizuki; Ando, Yoshinari; Kobayashi, Tohru; Matsuo, Yukari; Suzuki, Harukazu; Hayashizaki, Yoshihide; Kawai, Jun

    2010-04-01

    A novel method for the analysis of sequences of small RNAs using nucleotide triphosphates labeled with stable isotopes has been developed using time-of-flight mass spectroscopy combined with femtosecond laser ablation (fsLA-TOF-MS). Small RNAs synthesized with nucleotides enriched in 13C and 15N were efficiently atomized and ionized by single-shot fsLA and the isotope ratios 13C/12C and 15N/14N were evaluated using the TOF-MS method. By comparing the isotope ratios among four different configurations, the number of nucleotide contents of the control RNA sample were successfully reproduced.

  8. Enterococcus faecalis PrgJ, a VirB4-Like ATPase, Mediates pCF10 Conjugative Transfer through Substrate Binding

    OpenAIRE

    Li, Feng; Alvarez-Martinez, Cristina; Chen, Yuqing; Choi, Kyoung-Jae; Yeo, Hye-Jeong; Christie, Peter J.

    2012-01-01

    The Enterococcus faecalis prg and pcf genes of plasmid pCF10 encode a type IV secretion system (T4SS) required for conjugative transfer. PrgJ is a member of the VirB4 family of ATPases that are universally associated with T4SSs. Here, we report that purified PrgJ dimers displayed ATP binding and hydrolysis activities. A PrgJ nucleoside triphosphate (NTP) binding site mutation (K471E) slightly diminished ATP binding but abolished ATP hydrolysis in vitro and blocked pCF10 transfer in vivo. As s...

  9. Assay of covalent intermediate of 5'-nucleotide phosphodiesterase

    International Nuclear Information System (INIS)

    Blytt, H.J.; Brotherton, J.E.; Butler, L.

    1985-01-01

    A new procedure is reported for isolating a covalent phosphoryl enzyme (diester) intermediate of bovine intestinal 5'-nucleotide phosphodiesterase. The convenience of the procedure makes it possible to determine effects of reaction conditions on the yield of covalent intermediate. Under optimum conditions, using [methyl- 3 H]deoxythymidine 5'-triphosphate as substrate, more than 50% of the enzyme is recovered as thymidylyl enzyme, a 10-fold increase in yield over the previous procedure. Yields of thymidylyl enzyme were maximal at pH 4, whereas optimum catalytic activity is observed at pH greater than 9

  10. Generation of induced pluripotent stem cell (iPSC line from a 21-year-old X-linked adrenoleukodystrophy (X-ALD patient

    Directory of Open Access Journals (Sweden)

    Young Rang You

    2017-12-01

    Full Text Available X-linked Adrenoleukodystrophy (X-ALD is a genetic disease that caused by mutations in adenosine triphosphate [ATP]-binding-cassette transporter superfamily D member 1 (ABCD1 gene. We generated an induced pluripotent stem cell (iPSC line from a 21-year-old male X-ALD patient-derived fibroblasts by Sendai virus mediated reprogramming. Established iPSCs stably expanded while maintaining immunoreactivity for various pluripotency markers and alkaline phosphatase, as well as normal 44 + XY karyotype. Under the differentiation condition, the cells gave rise to cells of three germ layers.

  11. Investigation of protein binding of radiogallium. Progress report

    International Nuclear Information System (INIS)

    Hoffer, P.B.

    1985-01-01

    The main objective of the contract is to study aspects of the localization of gallium-67 in tumor are reported and compared with a new tumor imaging agent, specifically radiolabeled monoclonal tumor antibody. Our studies have demonstrated that phosphate compounds used in bone imaging do not significantly influence gallium-67 localization, that the adenosine triphosphate cannot be used to enhance gallium-67 uptake in malignant tissue, and that gallium-67 has higher affinity than radiolabeled anti-p97 for melanoma, even in tumors which produce significant amounts of p97 antigen on their cell surface. 23 refs., 17 figs., 5 tabs

  12. Phenothiazine-linked nucleosides and nucleotides for redox labelling of DNA

    Czech Academy of Sciences Publication Activity Database

    Simonova, Anna; Havran, Luděk; Pohl, Radek; Fojta, Miroslav; Hocek, Michal

    2017-01-01

    Roč. 15, č. 33 (2017), s. 6984-6996 ISSN 1477-0520 R&D Projects: GA ČR GBP206/12/G151 Grant - others:AV ČR(CZ) AP1501; AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae; Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 ; RVO:68081707 Keywords : electrochemical detection * primer extension * 2'-deoxyribonucleoside triphosphates Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.564, year: 2016 http://pubs.rsc.org/en/content/articlehtml/2017/ob/c7ob01439b

  13. One-Photon Absorption Properties from a Hybrid Polarizable Density Embedding/Complex Polarization Propagator Approach for Polarizable Solutions

    DEFF Research Database (Denmark)

    Hršak, Dalibor; Nørby, Morten Steen; Coriani, Sonia

    2018-01-01

    We present a formulation of the polarizable density embedding (PDE) method in combination with the complex polarization propagator (CPP) method for the calculation of absorption spectra of molecules in solutions. The method is particularly useful for the calculation of near-edge X-ray absorption...... fine structure (NEXAFS) spectra. We compare the performance of PDE-CPP with the previously formulated polarizable embedding (PE)-CPP model for the calculation of the NEXAFS spectra of adenine, formamide, glycine, and adenosine triphosphate (ATP) in water at the carbon and nitrogen K-edges, as well...

  14. DNA synthesis in permeabilized WI38 and MRC5 cells

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Carpenter, J.G.

    1980-01-01

    DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident

  15. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Suhadolnik, R.J.; Kariko, K.; Sobol, R.W. Jr.; Li, S.W.; Reichenbach, N.L.; Haley, B.E.

    1988-11-29

    The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.

  16. A review of nicotinamide: treatment of skin diseases and potential side effects.

    Science.gov (United States)

    Rolfe, Heidi M

    2014-12-01

    Nicotinamide, also known as niacinamide, is the amide form of vitamin B3. It is a precursor of essential coenzymes for numerous reactions in the body including adenosine triphosphate (ATP) production. Nicotinic acid, also known as niacin, is converted into nicotinamide in the body. The use of topical nicotinamide in the treatment of acne vulgaris; melasma; atopic dermatitis; rosacea; and oral nicotinamide in preventing nonmelanoma skin cancer is discussed. The possible side effects and consequences of excessive nicotinamide exposure are reviewed, including suggestions nicotinamide might have a role in the development of diabetes, Parkinson's disease, and liver damage. © 2014 Wiley Periodicals, Inc.

  17. Can isotopic exchange kinetics give valuable information on the rate of mineralization of organic phosphorus in soils ?

    OpenAIRE

    Frossard, E.; Lopez-Hernandez, D.; Brossard, Michel

    1996-01-01

    Bien que la minéralisation du phosphore organique puisse jouer un rôle important dans la nutrition des plantes, sa mesure a toujours été limitée du fait de problèmes méthodologiques. Notre objectif est de montrer si la détermination des cinétiques d'échange isotopiques peuvent donner des informations sur la minéralisation nette de composés organo-phosphatés dans les sols. En utilisant cette technique, la minéralisation de deux phosphates organiques (adénosine triphosphate, ATP, et nicotinamid...

  18. Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer

    Science.gov (United States)

    Kwok, Pui-Yan; Chen, Xiangning

    1999-01-01

    A method for detecting the presence of a target nucleotide or sequence of nucleotides in a nucleic acid is disclosed. The method is comprised of forming an oligonucleotide labeled with two fluorophores on the nucleic acid target site. The doubly labeled oligonucleotide is formed by addition of a singly labeled dideoxynucleoside triphosphate to a singly labeled polynucleotide or by ligation of two singly labeled polynucleotides. Detection of fluorescence resonance energy transfer upon denaturation indicates the presence of the target. Kits are also provided. The method is particularly applicable to genotyping.

  19. Enzymatic Ligation of Large Biomolecules to DNA

    DEFF Research Database (Denmark)

    Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David Henning

    2013-01-01

    application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other...... large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3′ hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations...

  20. Synthesis and biological evaluation of conformationally restricted adenine bicycloribonucleosides

    Czech Academy of Sciences Publication Activity Database

    Hřebabecký, Hubert; Procházková, Eliška; Šála, Michal; Plačková, Pavla; Tloušťová, Eva; Barauskas, O.; Lee, Y. J.; Tian, Y.; Mackman, R.; Nencka, Radim

    2015-01-01

    Roč. 13, č. 35 (2015), s. 9300-9313 ISSN 1477-0520 R&D Projects: GA ČR GPP207/12/P625; GA ČR GA15-09310S; GA ČR GA13-24880S; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : nucleic acid analogs * nucleoside triphosphates * sugar ring Subject RIV: CC - Organic Chemistry Impact factor: 3.559, year: 2015 http://pubs.rsc.org/en/content/articlepdf/2015/ob/c5ob00987a