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Sample records for ecto-nucleoside triphosphate diphosphohydrolase

  1. E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) of Leishmania amazonensis inhibits macrophage activation.

    PubMed

    Gomes, Rodrigo Saar; de Carvalho, Luana Cristina Faria; de Souza Vasconcellos, Raphael; Fietto, Juliana Lopes Rangel; Afonso, Luís Carlos Crocco

    2015-04-01

    Leishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis, is known for its ability to modulate the host immune response. Because a relationship between ectonucleotidase activity and the ability of Leishmania to generate injury in C57BL/6 mice has been demonstrated, in this study we evaluated the involvement of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of L. amazonensis in the process of infection of J774-macrophages. Our results show that high-activity parasites show increased survival rate in LPS/IFN-γ-activated cells, by inhibiting the host-cell NO production. Conversely, inhibition of E-NTPDase activity reduces the parasite survival rates, an effect associated with increased macrophage NO production. E-NTPDase activity generates substrate for the production of extracellular adenosine, which binds to A2B receptors and reduces IL-12 and TNF-α produced by activated macrophages, thus inhibiting NO production. These results indicate that E-NTPDase activity is important for survival of L. amazonensis within macrophages, showing the role of the enzyme in modulating macrophage response and lower NO production, which ultimately favors infection. Our results point to a new mechanism of L. amazonensis infection that may pave the way for the development of new treatments for this neglected disease. PMID:25554487

  2. Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

    PubMed Central

    Santos, Ramon F.; Pôssa, Marcela A. S.; Bastos, Matheus S.; Guedes, Paulo M. M.; Almeida, Márcia R.; DeMarco, Ricardo; Verjovski-Almeida, Sergio; Bahia, Maria T.; Fietto, Juliana L. R.

    2009-01-01

    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 candidates in chemotherapy

  3. Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities.

    PubMed

    Pinheiro, Carla M; Martins-Duarte, Erica S; Ferraro, Rodrigo B; Fonseca de Souza, André Luíz; Gomes, Marta T; Lopes, Angela H C S; Vannier-Santos, Marcos A; Santos, André L S; Meyer-Fernandes, José R

    2006-09-01

    The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages. PMID:16603157

  4. Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

    PubMed

    Brisevac, Dusica; Adzic, Marija; Laketa, Danijela; Parabucki, Ana; Milosevic, Milena; Lavrnja, Irena; Bjelobaba, Ivana; Sévigny, Jean; Kipp, Markus; Nedeljkovic, Nadezda

    2015-11-01

    Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis. PMID:26080748

  5. Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family.

    PubMed

    Braun, N; Fengler, S; Ebeling, C; Servos, J; Zimmermann, H

    2000-11-01

    We have isolated and characterized the cDNA encoding nucleoside triphosphate diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an open reading frame of 1365 bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971 Da and a predicted N-terminal hydrophobic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% and 51% identity respectively with sequences of the two related human and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transcripts with differing abundances. PCR analysis suggests a single open reading frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary (CHO) and PC12 cells for immunological analysis and protein isolation. The protein was contained in membrane fractions of transfected CHO cells and occurred in a soluble form in the cell culture supernatants. NTPDase6 preferentially hydrolysed nucleoside 5'-diphosphates. With different substrates the order of activity was GDP>IDP>UDP,CDP>ADP. Nucleoside 5'-triphosphates were hydrolysed only to a minor extent and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly and equally activated by Ca(2+) and Mg(2+) and had a K(m) for GDP of 211 microM. The immunohistochemical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is associated with the Golgi apparatus and to a small extent also with the plasma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of extracellular nucleotides. PMID:11042118

  6. Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis.

    PubMed

    de Souza, Ronny Francisco; Dos Santos, Yaro Luciolo; de Souza Vasconcellos, Raphael; Borges-Pereira, Lucas; Caldas, Ivo Santana; de Almeida, Márcia Rogéria; Bahia, Maria Terezinha; Fietto, Juliana Lopes Rangel

    2013-01-01

    Canine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leishmaniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI=92.60-100.0%) and a high specificity of 100% (95% CI=86.77-100.0%), with a high degree of confidence (k=1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis. PMID:23022017

  7. The Biochemical Properties of the Arabidopsis Ecto-Nucleoside Triphosphate Diphosphohydrolase AtAPY1 Contradict a Direct Role in Purinergic Signaling

    PubMed Central

    Massalski, Carolin; Bloch, Jeannine; Zebisch, Matthias; Steinebrunner, Iris

    2015-01-01

    The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three – GDP, IDP and UDP – were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca2+, Mg2+ and Mn2+ were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling

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

    PubMed Central

    Kiss, David S; Zsarnovszky, Attila; Horvath, Krisztina; Gyorffy, Andrea; Bartha, Tibor; Hazai, Diana; Sotonyi, Peter; Somogyi, Virag; Frenyo, Laszlo V; Diano, Sabrina

    2009-01-01

    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 mitochondrial respiration rates

  9. Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase.

    PubMed

    Weiss, Paulo Henrique Exterchoter; Batista, Franciane; Wagner, Glauber; Magalhães, Maria de Lourdes Borba; Miletti, Luiz Claudio

    2015-06-01

    Nucleoside triphosphate diphospho-hydrolases (NTPDases) catalyze the hydrolysis of several nucleosides tri and diphosphate playing major roles in eukaryotes including purinergic signaling, inflammation, hemostasis, purine salvage and host-pathogen interactions. These enzymes have been recently described in parasites where several evidences indicated their involvement in virulence and infection. Here, we have investigated the presence of NTPDase in the genome of Trypanosoma evansi. Based on the genomic sequence from Trypanosoma brucei, we have amplified an 1812 gene fragment corresponding to the T. evansi NTPDase gene. The protein was expressed in the soluble form and purified to homogeneity and enzymatic assays were performed confirming the enzyme identity. Kinetic parameters and substrate specificity were determined. The dependence of cations on enzymatic activity was investigated indicating the enzyme is stimulated by divalent cations and carbohydrates but inhibited by sodium. Bioinformatic analysis indicates the enzyme is a membrane bound protein facing the extracellular side of the cell with 98% identity to the T. brucei homologous NTPDase gene. PMID:25819299

  10. Structural Insight into Activation Mechanism of Toxoplasma gondii Nucleoside Triphosphate Diphosphohydrolases by Disulfide Reduction*

    PubMed Central

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

    2012-01-01

    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 Cys258–Cys268 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 Mg2+ 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 Arg492 and Glu493 in NTPDase1 by glycines in NTPDase3. PMID:22130673

  11. Crystal Structure of a Legionella pneumophila Ecto -Triphosphate Diphosphohydrolase, A Structural and Functional Homolog of the Eukaryotic NTPDases

    SciTech Connect

    Vivian, Julian P.; Riedmaier, Patrice; Ge, Honghua; Le Nours, Jérôme; Sansom, Fiona M.; Wilce, Matthew C.J.; Byres, Emma; Dias, Manisha; Schmidberger, Jason W.; Cowan, Peter J.; d'Apice, Anthony J.F.; Hartland, Elizabeth L.; Rossjohn, Jamie; Beddoe, Travis

    2010-04-19

    Many pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.

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

    PubMed

    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. PMID:18422631

  13. Structure-activity relationships of anthraquinone derivatives derived from bromaminic acid as inhibitors of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)

    PubMed Central

    Baqi, Younis; Weyler, Stefanie; Iqbal, Jamshed; Zimmermann, Herbert

    2008-01-01

    Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (Ki 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (Ki 0.328 μM) and NTPDase3 (Ki 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (Ki 1.5 μM). PMID:18528783

  14. Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

    PubMed

    Porcino, Gabriane Nascimento; Carvalho-Campos, Cristiane; Maia, Ana Carolina Ribeiro Gomes; Detoni, Michelle Lima; Faria-Pinto, Priscila; Coimbra, Elaine Soares; Marques, Marcos José; Juliano, Maria Aparecida; Juliano, Luiz; Diniz, Vanessa Álvaro; Corte-Real, Suzana; Vasconcelos, Eveline Gomes

    2012-10-01

    Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control. PMID:22921497

  15. Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function.

    PubMed

    Kauffenstein, Gilles; Pelletier, Julie; Lavoie, Elise G; Kukulski, Filip; Martín-Satué, Mireia; Dufresne, Sébastien S; Frenette, Jérôme; Ribas Fürstenau, Cristina; Sereda, Michal J; Toutain, Bertrand; Henrion, Daniel; Sullivan, Robert; Vial, Catherine; Sévigny, Jean

    2014-10-10

    In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αβMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials. PMID:25160621

  16. Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

    PubMed

    Handa, M; Guidotti, G

    1996-01-26

    A soluble ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (apyrase conserved regions) 1-4 that are highly conserved in known apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed. PMID:8579614

  17. Osmotic surveillance mediates rapid wound closure through nucleotide release

    PubMed Central

    Gault, William J.; Enyedi, Balázs

    2014-01-01

    Osmotic cues from the environment mediate rapid detection of epithelial breaches by leukocytes in larval zebrafish tail fins. Using intravital luminescence and fluorescence microscopy, we now show that osmolarity differences between the interstitial fluid and the external environment trigger ATP release at tail fin wounds to initiate rapid wound closure through long-range activation of basal epithelial cell motility. Extracellular nucleotide breakdown, at least in part mediated by ecto-nucleoside triphosphate diphosphohydrolase 3 (Entpd3), restricts the range and duration of osmotically induced cell migration after injury. Thus, in zebrafish larvae, wound repair is driven by an autoregulatory circuit that generates pro-migratory tissue signals as a function of environmental exposure of the inside of the tissue. PMID:25533845

  18. Hypercholesterolemia and Ecto-enzymes of Purinergic System: Effects of Paullinia cupana.

    PubMed

    Ruchel, J B; Rezer, J F P; Thorstenberg, M L; Dos Santos, C B; Cabral, F L; Lopes, S T A; da Silva, C B; Machado, A K; da Cruz, I B M; Schetinger, M R C; Gonçalves, J F; Leal, D B R

    2016-01-01

    Hypercholesterolemia is a metabolic disorder characterized by high levels of low-density lipoprotein and blood cholesterol, causing inflammatory lesion. Purinergic signaling modulates the inflammatory and immune responses through adenine nucleotides and nucleoside. Guaraná has hypocholesterolemic and antiinflammatory properties. Considering that there are few studies demonstrating the effects of guaraná powder on the metabolism of adenine nucleotides, we investigated its effects on the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase activity in lymphocytes of rats with diet-induced hypercholesterolemia. The rats were divided into hypercholesterolemic and normal diet groups. Each group was subdivided by treatment: saline, guaraná powder 12.5, 25, or 50 mg/kg/day and caffeine concentration equivalent to highest dose of guaraná, fed orally for 30 days. An increase in adenosine triphosphate hydrolysis was observed in the lymphocytes of rats with hypercholesterolemia and treated with 25 or 50 mg/kg/day when compared with the other groups. The hypercholesterolemic group treated with the highest concentration of guaraná powder showed decreased ecto-adenosine deaminase activity compared with the normal diet groups. Guaraná was able to reduce the total cholesterol and low-density lipoprotein cholesterol to basal levels in hypercholesterolemic rats. High concentrations of guaraná associated with a hypercholesterolemic diet are likely to have contributed to the reduction of the inflammatory process. PMID:26514663

  19. Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes.

    PubMed

    Eguchi, Ryota; Akao, Sanae; Otsuguro, Ken-ichi; Yamaguchi, Soichiro; Ito, Shigeo

    2015-05-01

    Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells. PMID:26003082

  20. Crystal structure of NTPDase2 in complex with the sulfoanthraquinone inhibitor PSB-071.

    PubMed

    Zebisch, Matthias; Baqi, Younis; Schäfer, Petra; Müller, Christa E; Sträter, Norbert

    2014-03-01

    In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis. PMID:24462745

  1. Ecto-nucleotidases distribution in human cyclic and postmenopausic endometrium.

    PubMed

    Aliagas, Elisabet; Vidal, August; Torrejón-Escribano, Benjamín; Taco, Maria del Rosario; Ponce, Jordi; de Aranda, Inmaculada Gómez; Sévigny, Jean; Condom, Enric; Martín-Satué, Mireia

    2013-06-01

    Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5'-nucleotidase (5'NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility. PMID:23225236

  2. Altered E-NTPDase/E-ADA activities and CD39 expression in platelets of sickle cell anemia patients.

    PubMed

    Castilhos, Lívia G; Doleski, Pedro H; Adefegha, Stephen A; Becker, Lara V; Ruchel, Jader B; Leal, Daniela B R

    2016-04-01

    Sickle cell anemia (SCA) is a hemoglobinopathy characterized by hemolysis and vaso-occlusions caused by rigidly distorted red blood cells. Sickle cell crisis is associated with extracellular release of nucleotides and platelets, which are critical mediators of hemostasis participating actively in purinergic thromboregulatory enzymes system.This study aimed to investigate the activities of purinergic system ecto-enzymes present on the platelet surface as well as CD39 and CD73 expressions on platelets of SCA treated patients. Fifteen SCA treated patients and 30 health subjects (control group) were selected. Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-5'-NT) and ecto-adenosine deaminase (E-ADA) activities were measured in platelets isolated from these individuals. Results demonstrated an increase of 41 % in the E-NTPDase for ATP hydrolysis, 52% for ADP hydrolysis and 60 % in the E-ADA activity in SCA patients (P<0.05); however, a two folds decrease in the CD39 expression in platelets was observed in the same group (P<0.01). The increased E-NTPDase activity could be a compensatory mechanism associated with the low expression of CD39 in platelets. Besides, alteration of these enzymes activities suggests that the purinergic system could be involved in the thromboregulatory process in SCA patients. PMID:27044834

  3. Optical Aptasensors for Adenosine Triphosphate

    PubMed Central

    Ng, Stella; Lim, Hui Si; Ma, Qian; Gao, Zhiqiang

    2016-01-01

    Nucleic acids are among the most researched and applied biomolecules. Their diverse two- and three-dimensional structures in conjunction with their robust chemistry and ease of manipulation provide a rare opportunity for sensor applications. Moreover, their high biocompatibility has seen them being used in the construction of in vivo assays. Various nucleic acid-based devices have been extensively studied as either the principal element in discrete molecule-like sensors or as the main component in the fabrication of sensing devices. The use of aptamers in sensors - aptasensors, in particular, has led to improvements in sensitivity, selectivity, and multiplexing capacity for a wide verity of analytes like proteins, nucleic acids, as well as small biomolecules such as glucose and adenosine triphosphate (ATP). This article reviews the progress in the use of aptamers as the principal component in sensors for optical detection of ATP with an emphasis on sensing mechanism, performance, and applications with some discussion on challenges and perspectives. PMID:27446501

  4. Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform.

    PubMed

    Rezende-Soares, F A; Carvalho-Campos, C; Marques, M J; Porcino, G N; Giarola, N L L; Costa, B L S; Taunay-Rodrigues, A; Faria-Pinto, P; Souza, M A; Diniz, V A; Corte-Real, S; Juliano, M A; Juliano, L; Vasconcelos, E G

    2010-04-01

    An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response. PMID:19961654

  5. Adenosine triphosphate inhibition of yeast trehalase.

    PubMed

    Panek, A D

    1969-09-01

    Yeast trehalase has been found to be inhibited non-competitively by adenosine triphosphate. Such a biological control could explain the accumulation of trehalose during the stationary phase of the growth curve. PMID:5370287

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

    PubMed

    Kowal, J M; Yegutkin, G G; Novak, I

    2015-12-01

    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 aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially 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 adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide

  7. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  8. Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis.

    PubMed

    Faria-Pinto, P; Meirelles, M N L; Lenzi, H L; Mota, E M; Penido, M L O; Coelho, P M Z; Vasconcelos, E G

    2006-09-01

    We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models. PMID:17308798

  9. Polyoxometalates--potent and selective ecto-nucleotidase inhibitors.

    PubMed

    Lee, Sang-Yong; Fiene, Amelie; Li, Wenjin; Hanck, Theodor; Brylev, Konstantin A; Fedorov, Vladimir E; Lecka, Joanna; Haider, Ali; Pietzsch, Hans-Jürgen; Zimmermann, Herbert; Sévigny, Jean; Kortz, Ulrich; Stephan, Holger; Müller, Christa E

    2015-01-15

    Polyoxometalates (POMs) are inorganic cluster metal complexes that possess versatile biological activities, including antibacterial, anticancer, antidiabetic, and antiviral effects. Their mechanisms of action at the molecular level are largely unknown. However, it has been suggested that the inhibition of several enzyme families (e.g., phosphatases, protein kinases or ecto-nucleotidases) by POMs may contribute to their pharmacological properties. Ecto-nucleotidases are cell membrane-bound or secreted glycoproteins involved in the hydrolysis of extracellular nucleotides thereby regulating purinergic (and pyrimidinergic) signaling. They comprise four distinct families: ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs), alkaline phosphatases (APs) and ecto-5'-nucleotidase (eN). In the present study, we evaluated the inhibitory potency of a series of polyoxometalates as well as chalcogenide hexarhenium cluster complexes at a broad range of ecto-nucleotidases. [Co4(H2O)2(PW9O34)2](10-) (5, PSB-POM142) was discovered to be the most potent inhibitor of human NTPDase1 described so far (Ki: 3.88 nM). Other investigated POMs selectively inhibited human NPP1, [TiW11CoO40](8-) (4, PSB-POM141, Ki: 1.46 nM) and [NaSb9W21O86](18-) (6, PSB-POM143, Ki: 4.98 nM) representing the most potent and selective human NPP1 inhibitors described to date. [NaP5W30O110](14-) (8, PSB-POM144) strongly inhibited NTPDase1-3 and NPP1 and may therefore be used as a pan-inhibitor to block ATP hydrolysis. The polyoxoanionic compounds displayed a non-competitive mechanism of inhibition of NPPs and eN, but appeared to be competitive inhibitors of TNAP. Future in vivo studies with selected inhibitors identified in the current study are warranted. PMID:25449596

  10. Hyperglycemia alters E-NTPDases, ecto-5'-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio).

    PubMed

    Capiotti, Katiucia Marques; Siebel, Anna Maria; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2016-06-01

    Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment. PMID:26769247

  11. The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    PubMed

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion. PMID:27482602

  12. CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection.

    PubMed

    Ennes-Vidal, V; Castro, R O S; Britto, C; Barrabin, H; D'Avila-Levy, C M; Moreira, O C

    2011-07-01

    Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis. PMID:21679488

  13. Characterization of circulating microparticle-associated CD39 family ecto-nucleotidases in human plasma.

    PubMed

    Jiang, Z Gordon; Wu, Yan; Csizmadia, Eva; Feldbrügge, Linda; Enjyoji, Keiichi; Tigges, John; Toxavidis, Vasilis; Stephan, Holger; Müller, Christa E; Müller, Christina E; McKnight, C James; Moss, Alan; Robson, Simon C

    2014-12-01

    Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism. PMID

  14. Chemoelectrical energy conversion of adenosine triphosphate

    NASA Astrophysics Data System (ADS)

    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.

  15. Efficient Synthesis of 5-Carboxy-2'-Deoxypyrimidine Nucleoside 5'-Triphosphates.

    PubMed

    Gong, Shan-Shan; Sun, Jian; You, Yue-Hai; Chen, Ji-Zong; Liu, Guo-Dong; Sun, Qi

    2016-06-01

    An efficient P(V)-N activation method for the synthesis of 5-carboxy-2'-deoxyuridine and 5-carboxy-2'-deoxycytidine triphosphates directly from the corresponding phosphoropiperidate precursors has been developed. PMID:27104859

  16. Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.

    PubMed

    Gollnest, Tristan; Dinis de Oliveira, Thiago; Rath, Anna; Hauber, Ilona; Schols, Dominique; Balzarini, Jan; Meier, Chris

    2016-04-18

    The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells. PMID:27008042

  17. Novel triphosphate phosphohydrolase activity of Clostridium thermocellum TTM, a member of the triphosphate tunnel metalloenzyme superfamily.

    PubMed

    Keppetipola, Niroshika; Jain, Ruchi; Shuman, Stewart

    2007-04-20

    Triphosphate tunnel metalloenzymes (TTMs) are a newly recognized superfamily of phosphotransferases defined by a unique active site residing within an eight-stranded beta barrel. The prototypical members are the eukaryal metal-dependent RNA triphosphatases, which catalyze the initial step in mRNA capping. Little is known about the activities and substrate specificities of the scores of TTM homologs present in bacterial and archaeal proteomes, nearly all of which are annotated as adenylate cyclases. Here we have conducted a biochemical and structure-function analysis of a TTM protein (CthTTM) from the bacterium Clostridium thermocellum. CthTTM is a metal-dependent tripolyphosphatase and nucleoside triphosphatase; it is not an adenylate cyclase. We have identified 11 conserved amino acids in the tunnel that are critical for tripolyphosphatase and ATPase activity. The most salient findings are that (i) CthTTM is 150-fold more active in cleaving tripolyphosphate than ATP and (ii) the substrate specificity of CthTTM can be transformed by a single mutation (K8A) that abolishes tripolyphosphatase activity while strongly stimulating ATP hydrolysis. Our results underscore the plasticity of CthTTM substrate choice and suggest how novel specificities within the TTM superfamily might evolve through changes in the residues that line the tunnel walls. PMID:17303560

  18. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages §...

  19. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages §...

  20. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages §...

  1. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages §...

  2. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Adenosine triphosphate release assay. 864.7040 Section 864.7040 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages §...

  3. Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.

    PubMed

    Zlatev, Ivan; Lackey, Jeremy G; Zhang, Ligang; Dell, Amy; McRae, Kathy; Shaikh, Sarfraz; Duncan, Richard G; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2013-02-01

    A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform. PMID:23260577

  4. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    PubMed

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  5. Modified Nucleoside Triphosphates for In-vitro Selection Techniques

    PubMed Central

    Dellafiore, María A.; Montserrat, Javier M.; Iribarren, Adolfo M.

    2016-01-01

    The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed. PMID:27200340

  6. Modified Nucleoside Triphosphates for In-vitro Selection Techniques.

    PubMed

    Dellafiore, María A; Montserrat, Javier M; Iribarren, Adolfo M

    2016-01-01

    The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed. PMID:27200340

  7. Modified Nucleoside Triphosphates for in-vitro Selection Techniques

    NASA Astrophysics Data System (ADS)

    Iribarren, Adolfo; Dellafiore, María; Montserrat, Javier

    2016-05-01

    The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

  8. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  9. A one-pot synthesis of α-l-threofuranosyl nucleoside triphosphates (tNTPs).

    PubMed

    Sau, Sujay P; Chaput, John C

    2016-07-15

    TNA (α-l-threofuranosyl nucleoside) triphosphates of adenosine (tATP), guanosine (tGTP), cytidine (tCTP), and thymidine (tTTP) were synthesized from their corresponding 3'-O-phosphoramidite derivatives using a novel one-pot reaction that is less moisture sensitive than traditional methods. The chemically synthesized tNTPs, despite containing an unnatural 3'-triphosphate moiety, are similar in thermal stability to natural nucleotide triphosphates. PMID:27246616

  10. An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5'-Triphosphates.

    PubMed

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

    2016-07-01

    A simple, reliable, and an efficient "one-pot, three step" chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5'-triphosphate (5-MeCTP), pseudouridine-5'-triphosphate (pseudoUTP) and N(1)-methylpseudouridine-5'-triphosphate (N(1)-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%). PMID:27159048

  11. Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide.

    PubMed

    Caiazzo, Elisabetta; Maione, Francesco; Morello, Silvana; Lapucci, Andrea; Paccosi, Sara; Steckel, Bodo; Lavecchia, Antonio; Parenti, Astrid; Iuvone, Teresa; Schrader, Jürgen; Ialenti, Armando; Cicala, Carla

    2016-07-15

    Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73

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

    SciTech Connect

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

    2006-11-01

    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.

  13. METABOLIC REGULATION OF ADENOSINE TRIPHOSPHATE SULFURYLASE IN YEAST

    PubMed Central

    de Vito, Peter C.; Dreyfuss, Jacques

    1964-01-01

    de Vito, Peter C. (Princeton University, Princeton, N.J.), and Jacques Dreyfuss. Metabolic regulation of adenosine triphosphate sulfurylase in yeast. J. Bacteriol. 88:1341–1348. 1964.—The metabolic regulation of adenosine triphosphate sulfurylase (ATP-sulfurylase) from baker's yeast was studied. The enzyme was strongly inhibited by low concentrations of adenosine-5′-phosphosulfate, 3′-phosphoadenosine-5′-phosphosulfate, and sulfide. Sulfide ion was a competitive inhibitor of ATP-sulfurylase. Cysteine, methionine, sulfite, and thiosulfate were not inhibitors of the enzyme. ATP-sulfurylase was repressed when yeast was grown in the presence of methionine, and derepressed when yeast was grown in the presence of cysteine. In contrast to these results, the enzyme sulfite reductase was repressed in cysteine-grown cells. Thus, the sulfate-reducing pathway in yeast appears to be regulated at its first step both by feedback inhibition (by sulfide) and by repression (by methionine). Other known controls in the cysteine biosynthetic pathway are discussed. PMID:14234791

  14. Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase.

    PubMed

    Salaemae, Wanisa; Yap, Min Y; Wegener, Kate L; Booker, Grant W; Wilce, Matthew C J; Polyak, Steven W

    2015-05-01

    Dethiobiotin synthetase (DTBS) plays a crucial role in biotin biosynthesis in microorganisms, fungi, and plants. Due to its importance in bacterial pathogenesis, and the absence of a human homologue, DTBS is a promising target for the development of new antibacterials desperately needed to combat antibiotic resistance. Here we report the first X-ray structure of DTBS from Mycobacterium tuberculosis (MtDTBS) bound to a nucleotide triphosphate (CTP). The nucleoside base is stabilized in its pocket through hydrogen-bonding interactions with the protein backbone, rather than amino acid side chains. This resulted in the unexpected finding that MtDTBS could utilise ATP, CTP, GTP, ITP, TTP, or UTP with similar Km and kcat values, although the enzyme had the highest affinity for CTP in competitive binding and surface plasmon resonance assays. This is in contrast to other DTBS homologues that preferentially bind ATP primarily through hydrogen-bonds between the purine base and the carboxamide side chain of a key asparagine. Mutational analysis performed alongside in silico experiments revealed a gate-keeper role for Asn175 in Escherichia coli DTBS that excludes binding of other nucleotide triphosphates. Here we provide evidence to show that MtDTBS has a broad nucleotide specificity due to the absence of the gate-keeper residue. PMID:25801336

  15. Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices

    PubMed Central

    Tabatadze, Nino; Woolley, Catherine

    2016-01-01

    Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.

  16. Ubiquitination and filamentous structure of cytidine triphosphate synthase.

    PubMed

    Pai, Li-Mei; Wang, Pei-Yu; Lin, Wei-Cheng; Chakraborty, Archan; Yeh, Chau-Ting; Lin, Yu-Hung

    2016-07-01

    Living organisms respond to nutrient availability by regulating the activity of metabolic enzymes. Therefore, the reversible post-translational modification of an enzyme is a common regulatory mechanism for energy conservation. Recently, cytidine-5'-triphosphate (CTP) synthase was discovered to form a filamentous structure that is evolutionarily conserved from flies to humans. Interestingly, induction of the formation of CTP synthase filament is responsive to starvation or glutamine depletion. However, the biological roles of this structure remain elusive. We have recently shown that ubiquitination regulates CTP synthase activity by promoting filament formation in Drosophila ovaries during endocycles. Intriguingly, although the ubiquitination process was required for filament formation induced by glutamine depletion, CTP synthase ubiquitination was found to be inversely correlated with filament formation in Drosophila and human cell lines. In this article, we discuss the putative dual roles of ubiquitination, as well as its physiological implications, in the regulation of CTP synthase structure. PMID:27116391

  17. Extraction and analysis of adenosine triphosphate from aquatic environments

    USGS Publications Warehouse

    Stephens, Doyle W.; Shultz, David J.

    1981-01-01

    A variety of adenosine triphosphate (ATP) extraction procedures have been investigated for their applicability to samples from aquatic environments. The cold sulfuric-oxalic acid procedure was best suited to samples consisting of water, periphyton, and sediments. Due to cation and fulvic acid interferences, a spike with a known quantity of ATP was necessary to estimate losses when sediments were extracted. Variable colonization densities for periphyton required that several replicates be extracted to characterize accurately the periphyton community. Extracted samples were stable at room temperature for one to five hours, depending on the ATP concentration, if the pH was below 2. Neutralized samples which were quick frozen and stored at -30C were stable for months. (USGS)

  18. Elevated synovial fluid concentration of adenosine triphosphate in dogs with osteoarthritis or sodium urate-induced synovitis of the stifle.

    PubMed

    Torres, Bryan T; Jimenez, David A; Budsberg, Steven C

    2016-07-19

    Adenosine triphosphate has been shown to stimulate nociceptive nerve terminals in joints. Elevated synovial fluid adenosine triphosphate concentrations as well as a correlation between synovial fluid adenosine triphosphate concentrations and osteoarthritic knee pain has been demonstrated in humans, but not yet in dogs. This study documented elevated synovial fluid adenosine triphosphate concentrations in the stifles of dogs with secondary osteoarthritis and urate-induced synovitis, as compared to normal stifles. PMID:27432274

  19. Identification of a nucleoside triphosphate binding site on calf thymus RNA polymerase II

    SciTech Connect

    Freund, E.; McGuire, P.M.

    1986-01-14

    A nucleoside triphosphate binding site on calf thymus RNA polymerase II was identified by using photoaffinity analogues of adenosine 5'-triphosphate and guanosine 5'-triphosphate. Both radiolabeled 8-azidoadenosine 5'-triphosphate (8-N3ATP) and radiolabeled 8-azidoguanosine 5'-triphosphate (8-N3GTP) bound to a single polypeptide of this enzyme. This polypeptide has a molecular mass of 37 kilodaltons and an isoelectric point of 5.4. Ultraviolet (UV) irradiation was necessary for photolabeling to occur. In addition, no labeling occurred when the probe was prephotolyzed or when the enzyme was inactivated. Furthermore, photolabeling of the enzyme could be decreased by preincubation with natural substrates. To provide evidence that the radiolabeled polypeptide forms a part of the domain of the nucleoside triphosphate binding site, experiments were performed using unlabeled 8-N3ATP. Although this unlabeled analogue was not a substrate for RNA polymerase II, it photoinactivated the enzyme in the presence of UV irradiation, and it inhibited transcription elongation by the enzyme in a competitive manner in the absence of UV irradiation. As in the case with photolabeling, photoinactivation by 8-N3ATP could be decreased by natural substrates; in both cases, purine ribonucleoside triphosphates were more efficient than pyrimidine nucleoside triphosphates. Furthermore, photoinactivation was saturable at about the same concentration as the inhibition constant for 8-N3ATP. Collectively, these results provide evidence that the radiolabeled polypeptide in calf thymus RNA polymerase II is an essential component for activity and suggest that this polypeptide may be part of this enzyme's purine ribonucleoside triphosphate binding site.

  20. Synthesis of α-l-Threofuranosyl Nucleoside Triphosphates (tNTPs)

    PubMed Central

    Zou, Keyong; Horhota, Allen; Yu, Biao; Szostak, Jack W.

    2005-01-01

    The α-l-threofuranosyl nucleoside triphosphates of T, G, and D (tTTP, tGTP, and tDTP) were synthesized from the described 2‘-O-DMT-protected derivatives using the Eckstein method, while the corresponding C derivative (tCTP) was prepared from the 2‘-O-acetyl derivative. The prepared α-l-threofuranosyl nucleoside triphosphates, despite being one carbon shorter than the native 2‘-deoxyfuranosyl nucleoside triphosphates, are effective substrates for selected DNA polymerases. PMID:15816733

  1. Inhibition of mammalian RNA polymerase by 5,6-dichlororibofuranosylbenzimidazole (DRB) and DRB triphosphate.

    PubMed Central

    Dreyer, C; Hausen, P

    1978-01-01

    DRB triphosphate inhibits activity of isolated RNA polymerase B, and, to a lesser extent, that of polymerase A. The same holds true for transcription in isolated nuclei. It does not act as an initiation inhibitor. In all cases, high concentrations of DRB triphosphate are required. Cells do not phosphorylate DRB to a measurable extent. hn RNA resistant to DRB is initiated with both ATP and GTP in the presence of the drug. These experiments render the hypothesis unlikely that DRB triphosphate in the cell specifically interferes with the initiation reaction of polymerase B. PMID:704359

  2. Formation of. beta. ,. gamma. -methylene-7,8-dihydroneopterin 3'-triphosphate from. beta. ,. gamma. -methyleneguanosine 5'-triphosphate by GTP cyclohydrolase I of Escherichia coli

    SciTech Connect

    Ferre, J.; Jacobson, K.B.

    1984-01-01

    GTP cyclohydrolase I of Escherichia coli converts (..beta..,..gamma..-methylene)GTP to a fluorescent product that is characterized as (..beta..,..gamma..-methylene)dihydroneopterin triphosphate. Interaction between the GTP analog and the enzyme gave a K/sub i/ of 3.0 ..mu..M, which may be compared to the K/sub m/ of 0.1 ..mu..M for GTP. This new analog of dihydroneopterin triphosphate may, in turn, be converted to the same greenish-yellow pteridines (compounds X, X1, and X2) that are obtained from dihydroneopterin triphosphate. Because of its stability to phosphatase action, this analog may be useful for studies in pteridine metabolism. 14 references, 5 figures.

  3. Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

    1975-01-01

    A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

  4. Oral sucrose for heel lance enhances adenosine triphosphate use in preterm neonates with respiratory distress

    PubMed Central

    Angeles, Danilyn M; Asmerom, Yayesh; Boskovic, Danilo S; Slater, Laurel; Bacot-Carter, Sharon; Bahjri, Khaled; Mukasa, Joseph; Holden, Megan; Fayard, Elba

    2015-01-01

    Objective: To examine the effects of oral sucrose on procedural pain, and on biochemical markers of adenosine triphosphate utilization and oxidative stress in preterm neonates with mild to moderate respiratory distress. Study design: Preterm neonates with a clinically required heel lance that met study criteria (n = 49) were randomized into three groups: (1) control (n = 24), (2) heel lance treated with placebo and non-nutritive sucking (n = 15) and (3) heel lance treated with sucrose and non-nutritive sucking (n = 10). Plasma markers of adenosine triphosphate degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile. Data were analyzed using repeated measures analysis of variance, chi-square and one-way analysis of variance. Results: We found that in preterm neonates who were intubated and/or were receiving ⩾30% FiO2, a single dose of oral sucrose given before a heel lance significantly increased markers of adenosine triphosphate use. Conclusion: We found that oral sucrose enhanced adenosine triphosphate use in neonates who were intubated and/or were receiving ⩾30% FiO2. Although oral sucrose decreased pain scores, our data suggest that it also increased energy use as evidenced by increased plasma markers of adenosine triphosphate utilization. These effects of sucrose, specifically the fructose component, on adenosine triphosphate metabolism warrant further investigation. PMID:26770807

  5. Adenosine triphosphate inhibits melatonin synthesis in the rat pineal gland.

    PubMed

    Souza-Teodoro, Luis Henrique; Dargenio-Garcia, Letícia; Petrilli-Lapa, Camila Lopes; Souza, Ewerton da Silva; Fernandes, Pedro A C M; Markus, Regina P; Ferreira, Zulma S

    2016-03-01

    Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance β-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by β-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects. PMID:26732366

  6. Behavior and stability of adenosine triphosphate (ATP) during chlorine disinfection.

    PubMed

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems. PMID:27295623

  7. Effect of calcium triphosphate cement on proximal humeral fracture osteosynthesis: a finite element analysis.

    PubMed

    Kennedy, Jim; Feerick, Emer; McGarry, Patrick; FitzPatrick, David; Mullett, Hannan

    2013-08-01

    PURPOSE. To measure the effect of void-filling calcium triphosphate cement on the loads at the implant-bone interface of a proximal humeral fracture osteosynthesis using a finite element analysis. METHODS. Finite element models of a 3-part proximal humeral fracture fixed with a plate with and without calcium triphosphate cement augmentation were generated from a quantitative computed tomography dataset of an intact proximal humerus. Material properties were assigned to bone fragments using published expressions relating Young's modulus to local Hounsfield number. Boundary conditions were then applied to the model to replicate the physiological loads. The effect of void-filling calcium triphosphate cement was analysed. RESULTS. When the void was filled with calcium triphosphate cement, the pressure gradient of the bone surrounding the screws in the medial fracture fragment decreased 97% from up to 21.41 to 0.66 MPa. Peak pressure of the fracture planes decreased 95% from 6.10 to 0.30 MPa and occurred along the medial aspect. The mean stress in the screw locking mechanisms decreased 78% from 71.23 to 15.92 MPa. The angled proximal metaphyseal screw had the highest stress. CONCLUSION. Augmentation with calcium triphosphate cement improves initial stability and reduces stress on the implant-bone interface. PMID:24014777

  8. Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes*

    PubMed Central

    Martinez, Jacobo; Truffault, Vincent; Hothorn, Michael

    2015-01-01

    Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family. PMID:26221030

  9. Theory of Polymer Entrapped Enzyme Ultramicroelectrodes: Application to Glucose and Adenosine Triphosphate Detection

    PubMed Central

    Kottke, Peter A.; Kranz, Christine; Kwon, Yong Koo; Masson, Jean-Francois; Mizaikoff, Boris; Fedorov, Andrei G.

    2010-01-01

    We validate, by comparison with experimental data, a theoretical description of the amperometric response of microbiosensors formed via enzyme entrapment. The utility of the theory is further illustrated with two relevant examples supported by experiments: (1) quantitative detection of glucose and (2) quantitative detection of adenosine triphosphate (ATP). PMID:20445817

  10. Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates

    PubMed Central

    Imoto, Shuhei; Patro, Jennifer N.; Jiang, Yu Lin; Oka, Natsuhisa; Greenberg, Marc M.

    2007-01-01

    The nucleoside triphosphates of N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (β-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and β-C-Fapy·dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3′-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of β-C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy·dGTP and β-C-Fapy·dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates. PMID:17090045

  11. A method of the rapid preparation of adenosine 5'-gamma-[32P] triphosphate by chemical synthesis.

    PubMed

    Koziołkiewicz, W; Pankowski, J; Janecka, A

    1978-01-01

    A new chemical method for the synthesis of adenosine 5'-gamma-[32P] triphosphate has been developed based on the reaction of adenosine 5'-diphosphate with ethyl chloroformate. The resulting active mixed anhydride was able to react with [32P]-triethylammonium orthophosphate to give gamma-[32P]ATP. PMID:219425

  12. Chemical synthesis of nucleoside-gamma-[32P]triphosphates of high specific activity.

    PubMed

    Janecka, A; Panusz, H; Pankowski, J; Koziołkiewicz, W

    1980-01-01

    A simple chemical procedure for the preparation of four common ribonucleoside 5-gamma-[32P]triphosphates of high specific activity (up to 10 Ci/mmole) based on the condensation of orthophosphoric acid with the corresponding nucleoside 5-diphosphate in the presence of ethyl chloroformate as well as the methods of purification and identification of the products are described. PMID:7375446

  13. Structural and functional characterization of a noncanonical nucleoside triphosphate pyrophosphatase from Thermotoga maritima

    SciTech Connect

    Awwad, Khaldeyah; Desai, Anna; Smith, Clyde; Sommerhalter, Monika

    2013-02-01

    A 2.15 Å resolution crystal structure of TM0159 with bound IMP and enzyme-kinetic data are presented. This noncanonical nucleoside triphosphatase from T. maritima helps to maintain a correct pool of DNA and RNA precursor molecules. The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k{sub cat}/K{sub m} values determined at 323 and 353 K fall between 1.31 × 10{sup 4} and 7.80 × 10{sup 4} M{sup −1} s{sup −1}. ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg{sup 2+} as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase)

  14. Structural and functional characterization of a noncanonical nucleoside triphosphate pyrophosphatase from Thermotoga maritima

    PubMed Central

    Awwad, Khaldeyah; Desai, Anna; Smith, Clyde; Sommerhalter, Monika

    2013-01-01

    The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k cat/K m values determined at 323 and 353 K fall between 1.31 × 104 and 7.80 × 104  M −1 s−1. ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg2+ as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase). PMID:23385455

  15. Synthesis and polymerase incorporation of β,γ-modified α-l-threofuranosyl thymine triphosphate mimics.

    PubMed

    Chen, Zhe; Meek, Kirsten N; Rangel, Alexandra E; Heemstra, Jennifer M

    2016-08-15

    Three β,γ-modified α-l-threofuranosyl nucleoside triphosphates were synthesized. The β,γ-modified tTTPs undergo a single incorporation event with HIV RT but undergo multiple incorporations to form full-length product with engineered thermophilic polymerases. PMID:27422338

  16. Multiple conformations of the nucleotide site of Kinesin family motors in the triphosphate state.

    PubMed

    Naber, Nariman; Larson, Adam; Rice, Sarah; Cooke, Roger; Pate, Edward

    2011-05-13

    Identifying conformational changes in kinesin family motors associated with nucleotide and microtubule (MT) binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide analog spin probes bound at the active sites of kinesin family motors to monitor conformational changes, we previously demonstrated that, in the ADP state, the open nucleotide site closes upon MT binding [Naber, N., Minehardt, T. J., Rice, S., Chen, X., Grammer, J., Matuska, M., et al. (2003). Closing of the nucleotide pocket of kinesin family motors upon binding to microtubules. Science, 300, 798-801]. We now extend these studies to kinesin-1 (K) and ncd (nonclaret disjunctional protein) motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin, with the mutation E236A (K-E236A; does not hydrolyze ATP) bound to ATP, show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the electron paramagnetic resonance spectrum, implying closing of the nucleotide site. The MT-bound states of all of the triphosphate analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have more restricted mobility than observed in MT-bound diphosphate states. Thus, the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and the electron microscopy of MT

  17. Multiple Conformations of the Nucleotide Site of Kinesin-Family Motors in the Triphosphate State

    PubMed Central

    Naber, Nariman; Larson, Adam; Rice, Sarah; Cooke, Roger; Pate, Edward

    2014-01-01

    Identifying conformational changes in kinesin-family motors associated with nucleotide and microtubule binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide-analog spin probes bound at the active sites of kinesin-family motors to monitor conformational changes, we previously demonstrated that in the ADP state the open nucleotide site closes upon microtubule (MT) binding1. We now extend these studies to kinesin-1 (K) and ncd motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin with the mutation E236A (K-E236A, does not hydrolyze ATP) bound to ATP show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate/analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the EPR spectrum, implying a closing of the nucleotide site. The MT-bound states of all of the triphosphate-analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have lower mobility than those observed in MT-bound diphosphate states. Thus the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and electron microscopy of MT-bound triphosphate analog states. PMID:21277856

  18. 6-Substituted 2-Aminopurine-2'-deoxyribonucleoside 5'-Triphosphates that Trace Cytosine Methylation.

    PubMed

    von Watzdorf, Janina; Marx, Andreas

    2016-08-17

    Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2'-deoxy 5-methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O(6) -alkylated 2'-deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6-substituted 2-aminopurine-2'-deoxyribonucleoside 5'-triphosphates modified at position 6 with various functionalities. We found that sensing of 5-methylation by this class of nucleotides is more general, not being restricted to O(6) -alkyl modification of dGTP but also applying to other functionalities. PMID:27253512

  19. Method for Adenosine 5′-Triphosphate Measurement on Coke Waste Activated Sludge

    PubMed Central

    Russell, James; Gauthier, Joseph J.

    1978-01-01

    Measurement of adenosine 5′-triphosphate (ATP) in coke waste activated sludge can provide a simple method for estimating the levels of viable microbes in the sludge. However, the presence of inhibitors such as phenol in the sludge interferes when the luciferin-luciferase method is used to measure ATP. These inhibiting substances can be removed from the sludge before extraction of ATP by washing the cells with dilute sodium dodecyl sulfate. PMID:16345281

  20. A nucleoside triphosphate-regulated, 3' exonucleolytic mechanism is involved in turnover of yeast mitochondrial RNAs.

    PubMed Central

    Min, J; Zassenhaus, H P

    1993-01-01

    We have employed cell-free transcription reactions with mitochondria isolated from Saccharomyces cerevisiae to study the mechanism of RNA turnover. The specificity of RNA turnover was preserved in these preparations, as were other RNA-processing reactions, including splicing, 3' end formation of mRNAs, and maturation of rRNAs. Turnover of nascent RNAs was found to occur exonucleolytically; endonucleolytic cleavage products were not detected during turnover of the omega intron RNA, which was studied in detail. However, these experiments still leave open the possibility that endonucleolytic cleavage products with very short half-lives are kinetic intermediates in the decay of omega RNA. Exonucleolytic turnover was regulated by nucleotide triphosphates and required their hydrolysis. A unique signature of this regulation was that any one of the eight standard ribo- or deoxyribonucleotide triphosphates supported RNA turnover. A novel hybrid selection protocol was used to determine the turnover rates of the 5', middle, and 3' portions of one mitochondrial transcript, the omega intron RNA. The results suggested that degradation along that transcript occurred with a 3'-->5' polarity. The similarity between features of mitochondrial RNA turnover and the properties of a nucleotide triphosphate-dependent 3' exoribonuclease that has been purified from yeast mitochondria suggests that this single enzyme is a key activity whose regulation is involved in the specificity of mitochondrial RNA turnover. Images PMID:7691792

  1. Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

    PubMed Central

    Toroney, Rebecca; Hull, Chelsea M.; Sokoloski, Joshua E.; Bevilacqua, Philip C.

    2012-01-01

    The protein kinase PKR is activated by RNA to phosphorylate eIF-2α, inhibiting translation initiation. Long dsRNA activates PKR via interactions with the dsRNA-binding domain (dsRBD). Weakly structured RNA also activates PKR and does so in a 5′-triphosphate (ppp)–dependent fashion, however relatively little is known about this pathway. We used a mutant T7 RNA polymerase to incorporate all four triphosphate-containing nucleotides into the first position of a largely single-stranded RNA and found absence of selectivity, in that all four transcripts activate PKR. Recognition of 5′-triphosphate, but not the nucleobase at the 5′-most position, makes this RNA-mediated innate immune response sensitive to a broad array of viruses. PKR was neither activated in the presence of γ-GTP nor recognized NTPs other than ATP in activation competition and ITC binding assays. This indicates that the binding site for ATP is selective, which contrasts with the site for the 5′ end of ppp-ssRNA. Activation experiments reveal that short dsRNAs compete with 5′-triphosphate RNAs and heparin for activation, and likewise gel-shift assays reveal that activating 5′-triphosphate RNAs and heparin compete with short dsRNAs for binding to PKR's dsRBD. The dsRBD thus plays a critical role in the activation of PKR by ppp-ssRNA and even heparin. At the same time, cross-linking experiments indicate that ppp-ssRNA interacts with PKR outside of the dsRBD as well. Overall, 5′-triphosphate-containing, weakly structured RNAs activate PKR via interactions with both the dsRBD and a distinct triphosphate binding site that lacks 5′-nucleobase specificity, allowing the innate immune response to provide broad-spectrum protection from pathogens. PMID:22912486

  2. Chloride permeability of rat brain membrane vesicles correlates with thiamine triphosphate content.

    PubMed

    Bettendorff, L; Hennuy, B; De Clerck, A; Wins, P

    1994-07-25

    Incubation of rat brain homogenates with thiamine or thiamine diphosphate (TDP) leads to a synthesis of thiamine triphosphate (TTP). In membrane vesicles subsequently prepared from the homogenates, increased TTP content correlates with increased 36Cl- uptake. A hyperbolic relationship was obtained with a K0.5 of 0.27 nmol TTP/mg protein. In crude mitochondrial fractions from the brains of animals previously treated with thiamine or sulbutiamine, a positive correlation between 36Cl- uptake and TTP content was found. These results, together with other results previously obtained with the patch-clamp technique, suggest that TTP is an activator of chloride channels having a large unit conductance. PMID:7953714

  3. A Small Aptamer with Strong and Specific Recognition of the Triphosphate of ATP

    PubMed Central

    Sazani, Peter L.; Larralde, Rosa

    2004-01-01

    We report the in vitro selection of an RNA-based ATP aptamer with the ability to discriminate between adenosine ligands based on their 5‘ phosphorylation state. Previous selection of ATP aptamers yielded molecules that do not significantly discriminate between ligands at the 5‘ position. By applying a selective pressure that demands recognition of the 5‘ triphosphate, we obtained an aptamer that binds to ATP with a Kd of approximately 5 μM, and to AMP with a Kd of approximately 5.5 mM, a difference of 1100-fold. This aptamer demonstrates the ability of small RNAs to interact with negatively charged moieties. PMID:15237981

  4. Fluorescent structural DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates.

    PubMed

    Anderson, Jon P; Reynolds, Bambi L; Baum, Kristin; Williams, John G

    2010-03-10

    Highly labeled DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates (dNTPs) were developed as a source of dNTPs for DNA polymerase. The particles were prepared by strand-displacement polymerization from a self-complementary circular template. Imaged by atomic force microscopy, these functionalized particles appear as condensed fuzzy balls with diameters between 50 and 150 nm. They emit a bright fluorescent signal, detected in 2 ms exposures with a signal-to-noise ratio of 25 when imaged using a TIR fluorescence microscope. PMID:20158249

  5. Enhanced Diffusion of Molecular Motors in the Presence of Adenosine Triphosphate and External Force

    NASA Astrophysics Data System (ADS)

    Shinagawa, Ryota; Sasaki, Kazuo

    2016-06-01

    The diffusion of a molecular motor in the presence of a constant external force is considered on the basis of a simple theoretical model. The motor is represented by a Brownian particle moving in a series of parabolic potentials placed periodically on a line, and the potential is switched stochastically from one parabola to another by a chemical reaction, which corresponds to the hydrolysis or synthesis of adenosine triphosphate (ATP) in motor proteins. It is found that the diffusion coefficient as a function of the force exhibits peaks. The mechanism of this diffusion enhancement and the possibility of observing it in F1-ATPase, a biological rotary motor, are discussed.

  6. Fluorescent Structural DNA Nanoballs Functionalized With Phosphate-Linked Nucleotide Triphosphates

    PubMed Central

    Anderson, Jon P.; Reynolds, Bambi L.; Baum, Kristin; Williams, John G.

    2010-01-01

    Highly labeled DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates (dNTPs) were developed as a source of dNTPs for DNA polymerase. The particles were prepared by strand-displacement polymerization from a self-complementary circular template. Imaged by atomic force microscopy, these functionalized particles appear as condensed fuzzy balls with diameters between 50–150 nm. They emit a bright fluorescent signal, detected in 2 msec exposures with a signal-to-noise of 25 when imaged using a TIR fluorescence microscope. PMID:20158249

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

    PubMed

    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. PMID:27261824

  8. Purification, Crystallization, and Preliminary Crystallographic Analysis of Deoxyuridine Triphosphate Nucleotidohydrolase from Arabidopsis Thaliana

    SciTech Connect

    Bajaj,M.; Moriyama, H.

    2007-01-01

    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 Angstroms resolution using Cu K{alpha} radiation. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 69.90, b = 70.86 Angstroms, c = 75.55 Angstroms . Assuming the presence of a trimer in the asymmetric unit, the solvent content was 30%, with a VM of 1.8 Angstroms 3 Da-1.

  9. Synthesis of Nucleoside Triphosphates from 2'-3'-Protected Nucleosides Using Trimetaphosphate.

    PubMed

    Mohamady, Samy; Taylor, Scott D

    2016-02-01

    Chemists have been attempting to triphosphorylate nucleosides and other alcohols using trimetaphosphate (TriMP) since the 1960s. However, this route appears to have been abandoned due to poor yields. The first practical syntheses of nucleoside triphosphates (NTPs) are reported using TriMP as the key reagent. This was achieved by reacting the tetrabutylammonium salt of TriMP with mesitylenesulfonyl chloride in the presence of DABCO in pyridine followed by the addition of an appropriately protected nucleoside and phthalimide. Quenching the reaction with aqueous buffer followed by hydrolysis of the OH protecting groups gave the NTPs in good yield. PMID:26759914

  10. Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts.

    PubMed

    Sillero, Maria A Günther; de Diego, Anabel; Tavares, Janeth E F; Silva, Joana A D Catanho da; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2009-08-15

    Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells. PMID:19414000

  11. Scope and Limitations of Typical Copper-Free Bioorthogonal Reactions with DNA: Reactive 2'-Deoxyuridine Triphosphates for Postsynthetic Labeling.

    PubMed

    Merkel, Marcus; Arndt, Stefanie; Ploschik, Damian; Cserép, Gergely B; Wenge, Ulrike; Kele, Péter; Wagenknecht, Hans-Achim

    2016-09-01

    Four triphosphates of 2'-deoxyuridine that carried the following bioorthogonally reactive groups were synthesized by organic-chemical methods. Two triphosphates with tetrazines and one with a cyclopropene moiety were designed for Diels-Alder reactions with inverse electron demand, and one triphosphate with a tetrazole core was designed for the "photoclick" cycloaddition. These triphosphates were not only successfully applied for oligonucleotide preparation by standard DNA polymerases, including Hemo KlenTaq, Vent, and Deep Vent, but also bypassed for full length primer extension products. Fluorescent labeling of the primer extension products was achieved by fluorophores with reactive counterparts and analyzed by polyacrylamide gel electrophoresis mobility shifts. The tetrazine-oligonucleotide conjugates were reacted with carboxymethylmonobenzocyclooctyne- and bicyclononyne-modified fluorophores. The yield of these postsynthetic reactions could significantly be improved by a more stable but still reactive nicotinic acid-derived tetrazine and by changing the key experimental conditions, mainly the pH of 7.2 and the temperature of 45-55 °C. The cyclopropene-oligonucleotide conjugate could be successfully labeled with a tetrazine-modified rhodamine in very good yields. The "photoclick" cycloaddition between tetrazole-oligonucleotide conjugates and a maleimide-modified dye worked quantitatively. The combination of primer extension, bypass, and bioorthogonal modification works also for double and triple labeling using the cyclopropene-modified 2'-deoxyuridine triphosphate. PMID:27513089

  12. Osteopontin stimulates gelsolin-associated phosphoinositide levels and phosphatidylinositol triphosphate-hydroxyl kinase.

    PubMed Central

    Chellaiah, M; Hruska, K

    1996-01-01

    Based on previous studies demonstrating activation of phosphatidylinositol 3-hydroxyl kinase (PI3-kinase) and stimulation of a change in cell shape, we examined the effect of osteopontin on the association of phospholipids with gelsolin, an actin-capping/severing protein. Osteopontin stimulated a rapid increase in phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate levels associated with gelsolin in Triton-soluble fractions of cell lysates. The increased levels of phosphatidylinositol triphosphate associated with gelsolin were due to stimulation of PI3-kinase activity associated with gelsolin in the Triton-soluble fractions, and they were blocked by the PI3-kinase inhibitor wortmannin. Osteopontin stimulated translocation of PI3-kinase from the Triton-insoluble to Triton-soluble gelsolin. Osteopontin also decreased Triton-soluble gelsolin/actin complexes consistent with actin uncapping, and increased F-actin levels, which were also blocked by wortmannin. The osteopontin effects were mediated through binding to the alpha(v)beta 3 integrin. Taken together, our studies indicate that osteopontin/alpha(v)beta 3-mediated changes in gelsolin-associated phosphoinositide levels and PI3-kinase activity are related to stimulation of F-actin formation in osteoclasts. Images PMID:8744948

  13. Systemic delivery of gemcitabine triphosphate via LCP nanoparticles for NSCLC and pancreatic cancer therapy.

    PubMed

    Zhang, Yuan; Kim, William Y; Huang, Leaf

    2013-04-01

    Nucleoside analogs are a significant class of anti-cancer agent. As prodrugs, they terminate the DNA synthesis upon transforming to their active triphosphate metabolites. We have encapsulated a biologically activate nucleotide analog (i.e. gemcitabine triphosphate (GTP)), instead of the nucleoside (i.e. gemcitabine) derivative, into a novel Lipid/Calcium/Phosphate nanoparticle (LCP) platform. The therapeutic efficacy of LCP-formulated GTP was evaluated in a panel of human non-small-cell lung cancer (NSCLC) and human pancreatic cancer models after systemic administrations. GTP-loaded LCPs induced cell death and arrested the cell cycle in the S phase. In vivo efficacy studies showed that intravenously injected GTP-loaded LCPs triggered effective apoptosis of tumor cells, significant reduction of tumor cell proliferation and cell cycle progression, leading to dramatic inhibition of tumor growth, with little in vivo toxicity. Broadly speaking, the current study offers preclinical proof-of-principle that many active nucleotide or phosphorylated nucleoside analogs could be encapsulated in the LCP nanoplatform and delivered systemically for a wide variety of therapeutic applications. PMID:23380359

  14. Systemic Delivery of Gemcitabine Triphosphate via LCP Nanoparticles for NSCLC and Pancreatic Cancer Therapy

    PubMed Central

    Zhang, Yuan; Kim, William Y; Huang, Leaf

    2013-01-01

    Nucleoside analogues are a significant class of anticancer agent. As prodrugs, they terminate the DNA synthesis upon transforming to their active triphosphate metabolites. We have encapsulated a biologically activate nucleotide analogue (i.e. gemcitabine triphosphate (GTP)), instead of the nucleoside (i.e. gemcitabine) derivative, into a novel Lipid/Calcium/Phosphate nanoparticle (LCP) platform. The therapeutic efficacy of LCP-formulated GTP was evaluated in a panel of human non-small-cell lung cancer (NSCLC) and human pancreatic cancer models after systemic administrations. GTP-loaded LCPs induced cell death and arrested the cell cycle in the S phase. In vivo efficacy studies showed that intravenously injected GTP-loaded LCPs triggered effective apoptosis of tumor cells, significant reduction of tumor cell proliferation and cell cycle progression, leading to dramatic inhibition of tumor growth, with little in vivo toxicity. Broadly speaking, the current study offers preclinical proof-of-principle that many active nucleotide or phosphorylated nucleoside analogues could be encapsulated in the LCP nanoplatform and delivered systemically for a wide variety of therapeutic applications. PMID:23380359

  15. Tracking the Dephosphorylation of Resveratrol Triphosphate in Skin by Confocal Raman Microscopy

    PubMed Central

    Zhang, Guojin; Flach, Carol R.; Mendelsohn, Richard

    2007-01-01

    Polyphenolic resveratrol has been identified as a potent antioxidant acting as both a free radical scavenger and an inhibitor of enzyme oxidative activity. However, the reactive propensity of resveratrol also limits its use in topical formulations. A transient derivative of resveratrol, resveratrol triphosphate, has been designed to provide a means for the delayed delivery of the active compound in skin tissue where endogenous enzymes capable of dephosphorylation reside. Confocal Raman microscopy studies of intact pigskin biopsies treated with modified resveratrol provided information about the spatial distribution and time-dependence of permeation and conversion to the native active form. Conversion to the active form was not observed when skin samples were exposed to steam, a procedure that likely inactivates endogenous skin enzymes. In addition, treatment with the triphosphate compared to the parent compound revealed a more homogeneous distribution of resveratrol throughout the stratum corneum and viable epidermis when the former was applied. Thus, the bioavailability of resveratrol in the epidermis appears to be enhanced upon application of the pro-molecule compared to resveratrol. PMID:17826862

  16. Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

    PubMed

    Bao, Jie; Ryu, Dewey D Y

    2005-02-20

    The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use. PMID:15643625

  17. Enzymatic assay for deoxyribonucleoside triphosphates using synthetic oligonucleotides as template primers

    SciTech Connect

    Sherman, P.A.; Fyfe, J.A. )

    1989-08-01

    The enzymatic assay for deoxyribonucleoside triphosphates has been improved by using synthetic oligonucleotides of a carefully defined sequence as template primers for DNA polymerase. High backgrounds, which limit the sensitivity of the assay when calf thymus DNA or alternating copolymers are used as template primers, were eliminated with these oligonucleotide template primers. Sensitivity was further increased by designing the template primer to incorporate multiple labeled deoxyribonucleotides per limiting unlabeled deoxyribonucleotide. Each of several DNA polymerases exhibited unique reaction characteristics with the oligonucleotide template primers, which was attributed to the differing exonuclease activities associated with these various enzymes. Assay optimization therefore included matching the polymerase with the template primer to obtain the lowest background reaction and highest sensitivity. This modified assay is particularly well suited for keeping cell sample size to a minimum in experimental protocols which generate large numbers of data points or require careful timing of sampling. With this technique, we measured the levels of all four deoxyribonucleoside triphosphates in extracts from as few as 2 x 10(4) cultured cells.

  18. Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases.

    PubMed

    Nikiforov, Theo T

    2014-01-01

    Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2',3'-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion. PMID:24096197

  19. Use of 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

    SciTech Connect

    Kohnken, R.E.; Mc Connell, D.G.

    1985-07-02

    In an in vitro incubation, 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate ( (gamma-/sup 32/P)-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with (gamma-/sup 32/P)-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, (gamma-/sup 32/P)-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by (gamma-/sup 32/P)-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.

  20. Low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa. Identification of ethanolamine triphosphate

    PubMed Central

    Drewry, David T.; Gray, George W.; Wilkinson, Stephen G.

    1972-01-01

    A careful examination of the low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa (N.C.T.C. 1999) revealed the presence of ethanolamine triphosphate. During storage, the compound decomposed to give ethanolamine pyrophosphate, identified in a previous study (Drewry et al., 1971); PPi may be a further decomposition product. Evidence for the attachment of ethanolamine triphosphate to a polysaccharide fraction was obtained, but the possibility that some was attached to the lipid A moiety was not excluded. Basic compounds released during the hydrolysis of lipopolysaccharide included amino acids, polyamines and oligopeptides. PMID:4632171

  1. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate.

    PubMed

    Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

    2014-04-25

    We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ∼100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm2 V(-1) s(-1). The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM. PMID:24671026

  2. Direct growth of graphene on quartz substrates for label-free detection of adenosine triphosphate

    NASA Astrophysics Data System (ADS)

    Xu, Shicai; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Yang, Cheng; Liu, Mei; Chen, Chuansong; Zhang, Chao

    2014-04-01

    We demonstrate that continuous, uniform graphene films can be directly synthesized on quartz substrates using a two-temperature-zone chemical vapor deposition system and that their layers can be controlled by adjusting the precursor partial pressure. Raman spectroscopy and transmission electron microscopy confirm the formation of monolayer graphene with a grain size of ˜100 nm. Hall measurements show a room-temperature carrier mobility above 1500 cm2 V-1 s-1. The optical transmittance and conductance of the graphene films are comparable to those of transferred metal-catalyzed graphene. The method avoids the complicated and skilled post-growth transfer process and allows the graphene to be directly incorporated into a fully functional biosensor for label-free detection of adenosine triphosphate (ATP). This device shows a fast response time of a few milliseconds and achieves a high sensitivity to ATP molecules over a very wide range from 0.002 to 5 mM.

  3. Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

    PubMed

    Hacker, Stephan M; Welter, Moritz; Marx, Andreas

    2015-01-01

    This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD. PMID:25754889

  4. Effect of cadmium on lake water bacteria as determined by the luciferase assay of adenosine triphosphate

    SciTech Connect

    Seyfried, P.L.; Horgan, C.B.L.

    1981-10-01

    A firefly luciferase assay of bacterial adenosine triphosphate (ATP) was developed to measure the toxic effects of cadmium ions on aquatic organisms. Toxicity was monitored using intracellular (I/C) ATP (in micrograms per litre) as well as plate counts (colony-forming units per millilitre). The bacteria, which belonged mainly to the families Enterobacteriaceae and Pseudomonadaceae, exhibited varying degrees of resistance to up to 100 ppm cadmium when grown in a glucose-salts medium at pH 6.8. Among the organisms tested, cadmium resistance decreased in the following order: Pseudomonas vesicularis > P. aeruginosa > Enterobacter sp. > P. fluorescens > Chromobacter sp. > Serratia sp. A rise in the pH of the growth medium from 5 to 7 resulted in increased toxicity of cadmium.

  5. Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

    PubMed Central

    Broyles, S S; Moss, B

    1987-01-01

    Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites. Images PMID:2437324

  6. Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals

    PubMed Central

    Gollnest, Tristan; de Oliveira, Thiago Dinis; Schols, Dominique; Balzarini, Jan; Meier, Chris

    2015-01-01

    The antiviral activity of nucleoside reverse transcriptase inhibitors is often limited by ineffective phosphorylation. We report on a nucleoside triphosphate (NTP) prodrug approach in which the γ-phosphate of NTPs is bioreversibly modified. A series of TriPPPro-compounds bearing two lipophilic masking units at the γ-phosphate and d4T as a nucleoside analogue are synthesized. Successful delivery of d4TTP is demonstrated in human CD4+ T-lymphocyte cell extracts by an enzyme-triggered mechanism with high selectivity. In antiviral assays, the compounds are potent inhibitors of HIV-1 and HIV-2 in CD4+ T-cell (CEM) cultures. Highly lipophilic acyl residues lead to higher membrane permeability that results in intracellular delivery of phosphorylated metabolites in thymidine kinase-deficient CEM/TK− cells with higher antiviral activity than the parent nucleoside. PMID:26503889

  7. Erythrocyte 2,3-diphosphoglycerate and adenosine-triphosphate in cretins living at high altitude.

    PubMed

    Adams, W H

    1976-01-01

    A comparison of concentrations of 2,3-diphosphoglycerate (2,3-DPG) and adenosine-triphosphate (ATP) in the red cells of cretins and normal controls living at 3,700 m in the Nepal Himalayas has shown that 2,3-DPG and ATP levels were higher in the cretins. A negative correlation between hemoglobin and 2.3-DPG level was found. Chronic hypoxia appears to have provided the additional stress required to differentiate the significance of thyroid hormone deficiency in producing anemia from its effect on 2,3-DPG levels. If thyroid hormone is in fact one regulator of 2,3-DPG, the anemia of hypothyroidism appears to be more significant. This also suggest that the anemia of hypothyroidism, is at least in part, "pathologic" as opposed to "adaptive". PMID:822672

  8. Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides.

    PubMed

    Zhao, Qiang; Lv, Qin; Wang, Hailin

    2015-08-15

    We previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMR-labeled aptamers enabled direct FA sensing ATP with a detection limit of 1 µM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets. PMID:25814408

  9. A sensitive LC-MS/MS method for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells.

    PubMed

    Tu, Xiaowei; Lu, Youming; Zhong, Dafang; Zhang, Yifan; Chen, Xiaoyan

    2014-08-01

    Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25-100 ng/10(7) cells with acceptable accuracy and precision using 50 μL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze-thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine. PMID:24529342

  10. RELATIONSHIP BETWEEN THE ATP (ADENOSINE TRIPHOSPHATE) CONTENT OF SUBSURFACE MATERIAL AND THE RATE OF BIODEGRADATION OF ALKYLBENZENES AND CHLOROBENZENE

    EPA Science Inventory

    The rate of biotransformation of toluene in unconsolidated subsurface material from sites at Lula, Oklahoma, USA and Conroe, Texas, USA was compared to the ATP (adenosine triphosphate) content of these materials. The rate of toluene degradation decreased with decreasing ATP conte...

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Association of Thymidylate Synthase Gene Polymorphisms with Stavudine Triphosphate Intracellular Levels and Lipodystrophy▿

    PubMed Central

    Domingo, Pere; Cabeza, M. Carmen; Pruvost, Alain; Torres, Ferran; Salazar, Juliana; del Mar Gutierrez, M.; Mateo, M. Gracia; Fontanet, Angels; Fernandez, Irene; Domingo, Joan C.; Villarroya, Francesc; Vidal, Francesc; Baiget, Montserrat

    2011-01-01

    The antiviral activity and toxicity of stavudine (d4T) depend on its triphosphate metabolite, stavudine triphosphate (d4T-TP). Therefore, modifications in intracellular levels of d4T-TP may change the toxicity profile of stavudine. d4T-TP intracellular levels in peripheral blood mononuclear cells were determined with a prominence liquid chromatograph connected to a triple-quadruple mass spectrometer. Polymorphisms in the thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), reduced folate carrier 1 (RFC1; SLC19A1), and cyclin D1 (CCND1) genes were determined by direct sequencing using an ABI Prism 3100 genetic analyzer or Fluidigm's Biomark system. The Mann-Whitney test, rank analysis of variance (with Bonferroni's adjusted post hoc comparisons), and logistic regression were used for the inferential analyses. Thirty-three stavudine-treated patients were enrolled in this cross-sectional study. d4T-TP intracellular levels were 11.50 fmol/106 cells (interquartile range [IQR] = 8.12 to 13.87 fmol/106 cells) in patients with a high-expression TS genotype (2/3G, 3C/3G, and 3G/3G), whereas in those with a low-expression TS genotype (2/2, 2/3C, and 3C/3C), they were 21.40 fmol/106 cells (IQR = 18.90 to 27.0 fmol/106 cells) (P < 0.0001). Polymorphisms in the MTHFR, DHFR, RFC1, and CCND1 genes did not influence the intracellular concentration of d4T-TP. d4T-TP levels were independently associated with the TS genotype (low versus high expression; odds ratio [OR] = 86.22; 95% confidence interval [CI] = 8.48 to nonestimable; P = 0.0023). The low-expression TS genotype was associated with the development of HIV/highly active antiretroviral therapy-associated lypodystrophy syndrome (HALS) (OR = 14.0; 95% CI = 2.09 to 108.0; P = 0.0032). Our preliminary data show that polymorphisms in the thymidylate synthase gene are strongly associated with d4T-TP intracellular levels and with development of HALS. PMID:21282454

  13. Association of thymidylate synthase gene polymorphisms with stavudine triphosphate intracellular levels and lipodystrophy.

    PubMed

    Domingo, Pere; Cabeza, M Carmen; Pruvost, Alain; Torres, Ferran; Salazar, Juliana; del Mar Gutierrez, M; Mateo, M Gracia; Fontanet, Angels; Fernandez, Irene; Domingo, Joan C; Villarroya, Francesc; Vidal, Francesc; Baiget, Montserrat

    2011-04-01

    The antiviral activity and toxicity of stavudine (d4T) depend on its triphosphate metabolite, stavudine triphosphate (d4T-TP). Therefore, modifications in intracellular levels of d4T-TP may change the toxicity profile of stavudine. d4T-TP intracellular levels in peripheral blood mononuclear cells were determined with a prominence liquid chromatograph connected to a triple-quadruple mass spectrometer. Polymorphisms in the thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), reduced folate carrier 1 (RFC1; SLC19A1), and cyclin D1 (CCND1) genes were determined by direct sequencing using an ABI Prism 3100 genetic analyzer or Fluidigm's Biomark system. The Mann-Whitney test, rank analysis of variance (with Bonferroni's adjusted post hoc comparisons), and logistic regression were used for the inferential analyses. Thirty-three stavudine-treated patients were enrolled in this cross-sectional study. d4T-TP intracellular levels were 11.50 fmol/10(6) cells (interquartile range [IQR] = 8.12 to 13.87 fmol/10(6) cells) in patients with a high-expression TS genotype (2/3G, 3C/3G, and 3G/3G), whereas in those with a low-expression TS genotype (2/2, 2/3C, and 3C/3C), they were 21.40 fmol/10(6) cells (IQR = 18.90 to 27.0 fmol/10(6) cells) (P < 0.0001). Polymorphisms in the MTHFR, DHFR, RFC1, and CCND1 genes did not influence the intracellular concentration of d4T-TP. d4T-TP levels were independently associated with the TS genotype (low versus high expression; odds ratio [OR] = 86.22; 95% confidence interval [CI] = 8.48 to nonestimable; P = 0.0023). The low-expression TS genotype was associated with the development of HIV/highly active antiretroviral therapy-associated lypodystrophy syndrome (HALS) (OR = 14.0; 95% CI = 2.09 to 108.0; P = 0.0032). Our preliminary data show that polymorphisms in the thymidylate synthase gene are strongly associated with d4T-TP intracellular levels and with development of HALS. PMID:21282454

  14. The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit.

    PubMed Central

    Ferenczi, M A; Homsher, E; Trentham, D R

    1984-01-01

    The time course of magnesium adenosine triphosphate (Mg ATP) cleavage in chemically skinned muscle fibres of the rabbit was measured by a method in which Mg ATP cleavage was initiated by photolytic release of ATP from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and terminated by rapid freezing 50 ms to 8 s later. Up to 5 mM-ATP was released following a single 50 ns laser pulse at 347 nm. Mg ATP cleavage was measured at 19 degrees C in the presence and absence of calcium ions, for fibres near rest length and stretched beyond overlap of the myofilaments. At full overlap and in the absence of calcium (less than 10(-8) M) and nucleotide, the fibres developed rigor tension. Following the laser pulse the tension decreased to that of a relaxed fibre in two distinct phases. The first phase lasted about 40 ms and was followed by a second phase during which tension decreased to zero with an approximately exponential time course with a rate constant of 11 s-1. In the presence of 2 X 10(-5) M-free calcium ions, the initial phase following the laser flash lasted approximately 13 ms, and was followed by an exponential rise of tension with a rate constant of 28 s-1. The active tension reached by the muscle fibres was 54 kN/m2. For fibres stretched beyond overlap, no change in tension was observed following the release of Mg ATP. Under all conditions the time course of Mg ATP cleavage was biphasic, and consisted of a rapid initial burst of ADP formation, complete within 50 ms, followed by a slower steady-state rate of Mg ATP cleavage. The number of molecules of Mg ATP cleaved during the burst was approximately equal to the number of myosin subfragment 1 heads for fibres at full myofilament overlap, and equal to 0.7 molecules per myosin subfragment 1 head for fibres stretched beyond overlap. At full overlap in the presence of calcium ions, the steady-state rate equalled 1.8 mol Mg ATP cleaved per mole myosin subfragment 1 head per second. In all other cases the

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

    PubMed

    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. PMID:26838432

  16. Galactosylated Chitosan Oligosaccharide Nanoparticles for Hepatocellular Carcinoma Cell-Targeted Delivery of Adenosine Triphosphate

    PubMed Central

    Zhu, Xiu Liang; Du, Yong Zhong; Yu, Ri Sheng; Liu, Ping; Shi, Dan; Chen, Ying; Wang, Ying; Huang, Fang Fang

    2013-01-01

    Nanoparticles composed of galactosylated chitosan oligosaccharide (Gal-CSO) and adenosine triphosphate (ATP) were prepared for hepatocellular carcinoma cell-specific uptake, and the characteristics of Gal-CSO/ATP nanoparticles were evaluated. CSO/ATP nanoparticles were prepared as a control. The average diameter and zeta potential of Gal-CSO/ATP nanoparticles were 51.03 ± 3.26 nm and 30.50 ± 1.25 mV, respectively, suggesting suitable properties for a drug delivery system. Subsequently, the cytotoxicity of Gal-CSO/ATP nanoparticles were examined by the methyl tetrazolium (MTT) assay, and the half maximal inhibitory concentration (IC50) values were calculated with HepG2 (human hepatocellular carcinoma cell line) cells. The results showed that the cytotoxic effect of nanoparticles on HepG2 cells was low. In the meantime, it was also found that the Gal-CSO/ATP nanoparticles could be uptaken by HepG2 cells, due to expression of the asialoglycoprotein receptor (ASGP-R) on their surfaces. The presented results indicate that the Gal-CSO nanoparticles might be very attractive to be used as an intracellular drug delivery carrier for hepatocellular carcinoma cell targeting, thus warranting further in vivo or clinical investigations. PMID:23899789

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

    NASA Technical Reports Server (NTRS)

    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.

  18. Ribonucleoside Triphosphates as Substrate of Human Immunodeficiency Virus Type 1 Reverse Transcriptase in Human Macrophages*

    PubMed Central

    Kennedy, Edward M.; Gavegnano, Christina; Nguyen, Laura; Slater, Rebecca; Lucas, Amanda; Fromentin, Emilie; Schinazi, Raymond F.; Kim, Baek

    2010-01-01

    We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4+ T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22–320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3′-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3′-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4+ T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection. PMID:20924117

  19. Fullerene derived molecularly imprinted polymer for chemosensing of adenosine-5'-triphosphate (ATP).

    PubMed

    Sharma, Piyush S; Dabrowski, Marcin; Noworyta, Krzysztof; Huynh, Tan-Phat; Kc, Chandra B; Sobczak, Janusz W; Pieta, Piotr; D'Souza, Francis; Kutner, Wlodzimierz

    2014-09-24

    For molecular imprinting of oxidatively electroactive analytes by electropolymerization, we used herein reductively electroactive functional monomers. As a proof of concept, we applied C60 fullerene adducts as such for the first time. For that, we derivatized C60 to bear either an uracil or an amide, or a carboxy addend for recognition of the adenosine-5'-triphosphate (ATP) oxidizable analyte with the ATP-templated molecularly imprinted polymer (MIP-ATP). Accordingly, the ATP complex with all of the functional monomers formed in solution was potentiodynamically electropolymerized to deposit an MIP-ATP film either on an Au electrode of the quartz crystal resonator or on a Pt disk electrode for the piezoelectric microgravimetry (PM) or capacitive impedimetry (CI) determination of ATP, respectively, under the flow-injection analysis (FIA) conditions. The apparent imprinting factor for ATP was ∼4.0. After extraction of the ATP template, analytical performance of the resulting chemosensors, including detectability, sensitivity, and selectivity, was characterized. The limit of detection was 0.3 and 0.03mM ATP for the PM and CI chemosensor, respectively. The MIP-ATP film discriminated structural analogues of ATP quite well. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the experimental data of the ATP sorption and sorption stability constants appeared to be nearly independent of the adopted sorption model. PMID:25172817

  20. 2-Aminoethoxydiphenyl borate, a inositol 1,4,5-triphosphate receptor inhibitor, prevents atrial fibrillation.

    PubMed

    Xiao, Junjie; Liang, Dandan; Zhao, Hong; Liu, Ying; Zhang, Hong; Lu, Xiaowei; Liu, Yi; Li, Jun; Peng, Luying; Chen, Yi-Han

    2010-07-01

    The expression of the inositol 1,4,5-triphosphate receptor (IP3R) is upregulated and the function of IP3R also increases during atrial fibrillation (AF). 2-Aminoethoxydiphenyl borate (2-APB) is a membrane-permeable inhibitor of IP3R. However, the effect of 2-APB on AF is unknown. The aim of the present study is to explore the effects of 2-APB on AF. In vitro rabbit heart models of ischemia-, stretch- and cholinergic agitation-induced AF were developed. Fura-2-acetoxymethyl (Fura-2-AM) and Mg2+-Fura-2-AM were used to monitor alterations of intracellular Ca2+ and ATP, respectively, in HL-1 cells, an atrial muscle cell line, under chemical ischemia or cholinergic agitation. The results showed that inhibition of IP3R significantly reduced the incidence and its probability of being sustained in all three types of AF. IP3R inhibition ameliorated the cytoplasmic Ca2+ overload and energy compromise resulting from chemical ischemia or cholinergic agitation. Thus, IP3R inhibition may be a novel target for AF treatment, and IP3R may be an important molecule in the context of different kinds of AF. PMID:20472714

  1. Adenosine Triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells.

    PubMed

    Cutarelli, Alessandro; Marini, Mario; Tancredi, Virginia; D'Arcangelo, Giovanna; Murdocca, Michela; Frank, Claudio; Tarantino, Umberto

    2016-05-01

    In the last years adenosine triphosphate (ATP) and subsequent purinergic system activation through P2 receptors were investigated highlighting their pivotal role in bone tissue biology. In osteoblasts ATP can regulate several activities like cell proliferation, cell death, cell differentiation and matrix mineralization. Since controversial results exist, in this study we analyzed the ATP effects on differentiation and mineralization in human osteoblast-like Saos-2 cells. We showed for the first time the altered functional activity of ATP receptors. Despite that, we found that ATP can reduce cell proliferation and stimulate osteogenic differentiation mainly in the early stages of in vitro maturation as evidenced by the enhanced expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and Osteocalcin (OC) genes and by the increased ALP activity. Moreover, we found that ATP can affect mineralization in a biphasic manner, at low concentrations ATP always increases mineral deposition while at high concentrations it always reduces mineral deposition. In conclusion, we show the osteogenic effect of ATP on both early and late stage activities like differentiation and mineralization, for the first time in human osteoblastic cells. PMID:27189526

  2. Adenosine triphosphate quantification as related to cryptobiosis, nematode eggs, and larvae.

    PubMed

    Spurr, H W

    1976-04-01

    Sonification was the most effective method used for disintegrating nematode eggs and larvae for adenosine triphosphate (ATP) determinations. Sensitivity of the assay was sufficient to measure ATP in one larva. Second-stage larvae of Anguina tritici averaged 1 x 10 femtograms (fg) ATP and Meloidogyne incognita eggs, 0.8 x 105 fg ATP. Larvae of Panagrellus redivivus, a saprobe, averaged 12.2 x 105 fg ATP, a measurement which was considerably higher than the ATP levels in plant parasites. Endophytic bacteria and fungi from wheat galls were detected as background organisms associated with A. tritiei activated by hydration. Also, bacteria in suspensions of eggs from M. incognita prepared with NaCIO were measured by the use of butanol extraction and ATP determination. Second-stage A. tritici larvae increased in ATP content within 40 min after being activated from cryptobiosis by hydration. In the cryptobiotic state, larvae had 50% less ATP than when active. ATP concentrations were similar in galls of different ages. Apparently, ATP concentrations do not change during cryptobiosis. Starvation results in a decline in ATP concentration/larva. Subjecting A. tritici larvae to the lethal temperature of 60 C resulted in a three-fold increase in the decay rate of ATP over that of larvae sonified, then heated at 60 C. These results suggest an association between ATP decay and the mechanism that causes death of larvae at elevated temperatures. PMID:19308214

  3. Detection of adenosine 5'-triphosphate by fluorescence variation of oligonucleotide-templated silver nanoclusters.

    PubMed

    Lee, Jennifer Daneen; Cang, Jinshun; Chen, Ying-Chieh; Chen, Wei-Yu; Ou, Chung-Mao; Chang, Huan-Tsung

    2014-08-15

    Oligonucleotide-templated Ag nanoclusters (DNA-Ag NCs) prepared from AgNO3 using an oligonucleotide (5'-TAACCCCTAACCCCT-3') as a template and NaBH4 as a reducing agent have been used for sensing of adenosine 5'-triphosphate (ATP). The fluorescence intensity and emission wavelength of DNA-Ag NCs are dependent on the pH value and ATP concentration. At pH 3.0 and 11.0, ATP shows greater effects on fluorescence of the DNA-Ag NCs. Upon increasing ATP concentration from 10 to 50μM, their emission wavelength at pH 3.0 shifts from 525 to 585nm. At pH 11.0, their fluorescence intensity (510nm) increases upon increasing ATP concentration. The circular dichroism (CD), electrospray ionization-mass spectrometry (ESI-MS), absorption, and fluorescence results indicate that ATP and pH affect the interactions between DNAs and Ag atoms, resulting in changes in their fluorescence. The DNA-Ag NCs allow detection of ATP over a concentration range of 0.1-10μM, with a limit of detection 33nM. Practicality of the DNA-Ag NCs probe has been validated with the determination of ATP concentrations in the lysate of MDA-MB-231 breast carcinoma cells. PMID:24657647

  4. Leptin suppresses adenosine triphosphate-induced impairment of spinal cord astrocytes.

    PubMed

    Li, Baoman; Qi, Shuang; Sun, Guangfeng; Yang, Li; Han, Jidong; Zhu, Yue; Xia, Maosheng

    2016-10-01

    Spinal cord injury (SCI) causes long-term disability and has no clinically effective treatment. After SCI, adenosine triphosphate (ATP) may be released from neuronal cells and astrocytes in large amounts. Our previous studies have shown that the extracellular release of ATP increases the phosphorylation of cytosolic phospholipase A2 (cPLA2 ) and triggers the rapid release of arachidonic acid (AA) and prostaglandin E2 (PGE2) via the stimulation of epidermal growth factor receptor (EGFR) and the downstream phosphorylation of extracellular-regulated protein kinases 1 and 2. Leptin, a glycoprotein, induces the activation of the Janus kinase (JAK2)/signal transducers and activators of transcription-3 (Stat3) pathway via the leptin receptor. In this study, we found that 1) prolonged leptin treatment suppressed the ATP-stimulated release of AA and PGE2 from cultured spinal cord astrocytes; 2) leptin elevated the expression of caveolin-1 (Cav-1) via the JAK2/Stat3 signaling pathway; 3) Cav-1 blocked the interaction between Src and EGFR, thereby inhibiting the phosphorylation of EGFR and cPLA2 and attenuating the release of AA or PGE2; 4) pretreatment with leptin decreased ;he level of apoptosis and the release of interleukin-6 from cocultured neurons and astrocytes; and 5) leptin improved the recovery of locomotion in mice after SCI. Our results highlight leptin as a promising therapeutic agent for SCI. © 2016 Wiley Periodicals, Inc. PMID:27316329

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

    PubMed

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

    2015-01-01

    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 × 10(6) 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%. PMID:26638983

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

    DOE PAGESBeta

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; et al

    2015-03-23

    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 proteinmore » 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.« less

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

    SciTech Connect

    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

    2015-03-23

    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.

  8. Adenosine 5'-triphosphate release evoked by electrical nerve stimulation from the guinea-pig gallbladder.

    PubMed

    Takahashi, T; Kusunoki, M; Ishikawa, Y; Kantoh, M; Yamamura, T; Utsunomiya, J

    1987-01-28

    The endogenous release of adenosine 5'-triphosphate (ATP) from strips of guinea-pig gallbladder during transmural stimulation (TS) was measured with a firefly luciferine-luciferase reaction. TS (15V, 1 ms, 0.5-5 Hz, for 1 min) caused a rapid and marked increase of ATP release in a frequency-dependent manner. Both ATP release and contractions evoked by TS (15 V, 5 Hz, 1 ms) were completely abolished in Ca-free medium. BaCl2 (3 X 10(-3) M), a direct muscle stimulant, produced almost the same degree of contractile tension as TS (15 V, 5 Hz, 1 ms) while the ATP release induced by BaCl2 was significantly reduced to about 60 percent of that induced by TS. Atropine (10(-6) M) significantly reduced TS-evoked contraction without affecting ATP release. It was suggested, therefore, that some of the ATP release induced by TS was of neural origin. Theophylline (a P1-purinoreceptor antagonist) 10(-6) M, quinidine (a non-specific P2-purinoreceptor antagonist) 10(-6) M and apamin (a potassium channel blocking agent) 10(-8) M had no effects on TS-evoked contraction and ATP release, suggesting the absence of a presynaptic autoregulatory mechanism of ATP release in the guinea-pig gallbladder. PMID:3556400

  9. Production and utilization of dissolved adenosine 5'-triphosphate in marine and freshwater ecosystems

    SciTech Connect

    Peele, E.R.

    1985-01-01

    Concentrations of dissolved adenosine triphosphate (DATP) were influenced primarily by in situ biological processes such as uptake by heterotrophic microorganisms and release by either phytoplankton or zooplankton or through zooplankton-phytoplankton interactions. Rapid turnover of dissolved ATP via uptake by bacterioplankton was observed in an estuary (Sapelo Island, Georgia) and two freshwater lakes (Lake Oglethorpe, Georgia and Lago Lake, Georgia). Turnover times for DATP, based on microbial assimilation of (/sup 3/H)ATP, were on the order of hours to days in all three environments. DATP was not taken up intact by the natural microbial populations; rather, it was degraded to adenine, ribose and inorganic phosphate prior to or during transport. The primary mechanism for DATP uptake was via dephosphorylation of the nucleotide and cleavage of resultant nucleoside to adenine and ribose which then entered the cells by separate transport systems. The rate of DATP assimilation by freshwater microorganisms varied markedly-over a diel cycle. Results from microcosm experiments in which the authors compared the rates of DATP release by phytoplankton (Chlamydomonas sp.) alone, zooplankton (Daphnia sp.) alone or phytoplankton and zooplankton together under feeding conditions supported those hypotheses. Net extracellular release of DATP by Chlamydomonas was negligible in short-term experiments, and in systems containing both Daphnia and Chlamydomonas, changes in DATP over time were approximately 3-fold greater than the sum of changes observed in systems containing either organism alone.

  10. A Graphene and Aptamer Based Liquid Gated FET-Like Electrochemical Biosensor to Detect Adenosine Triphosphate.

    PubMed

    Mukherjee, Souvik; Meshik, Xenia; Choi, Min; Farid, Sidra; Datta, Debopam; Lan, Yi; Poduri, Shripriya; Sarkar, Ketaki; Baterdene, Undarmaa; Huang, Ching-En; Wang, Yung Yu; Burke, Peter; Dutta, Mitra; Stroscio, Michael A

    2015-12-01

    Here we report successful demonstration of a FET-like electrochemical nano-biosensor to accurately detect ultralow concentrations of adenosine triphosphate. As a 2D material, graphene is a promising candidate due to its large surface area, biocompatibility, and demonstrated surface binding chemistries and has been employed as the conducting channel. A short 20-base DNA aptamer is used as the sensing element to ensure that the interaction between the analyte and the aptamer occurs within the Debye length of the electrolyte (PBS). Significant increase in the drain current with progressive addition of ATP is observed whereas for control experiments, no distinct change in the drain current occurs. The sensor is found to be highly sensitive in the nanomolar (nM) to micromolar ( μM) range with a high sensitivity of 2.55 μA (mM) (-1), a detection limit as low as 10 pM, and it has potential application in medical and biological settings to detect low traces of ATP. This simplistic design strategy can be further extended to efficiently detect a broad range of other target analytes. PMID:26595926

  11. Genetics and complementation of Haemophilus influenzae mutants deficient in adenosine 5'-triphosphate-dependent nuclease.

    PubMed Central

    Kooistra, J; Small, G D; Setlow, J K; Shapanka, R

    1976-01-01

    Eight different mutations in Haemophilus influenzae leading to deficiency in adenosine 5'-triphosphate (ATP)-dependent nuclease have been investigated in strains in which the mutations of the originally mutagenized strains have been transferred into the wild type. Sensitivity to mitomycin C and deoxycholate and complementation between extracts and deoxyribonucleic acid (DNA)-dependent ATPase activity have been measured. Genetic crosses have provided information on the relative position of the mutations on the genome. There are three complementation groups, corresponding to three genetic groups. The strains most sensitive to mitomycin and deoxycholate, derived from mutants originally selected on the basis of sensitivity to mitomycin C or methyl methanesulfonate, are in one group. Apparently all these sensitive strains lack DNA-dependent ATPase activity, as does a strain intermediate in sensitivity to deoxycholate, which is the sole representative of another group. There are four strains that are relatively resistant to deoxycholate and mitomycin C, and all of these contain the ATPase activity. Three of these are in the same genetic and complementation group, whereas the other incongruously belongs in the same group as the sensitive strains. It is postulated that there are three cistrons in H. influenzae that code for the three known subunits of the ATP-dependent nuclease. PMID:177397

  12. Reexamination of magnetic isotope and field effects on adenosine triphosphate production by creatine kinase

    PubMed Central

    Crotty, Darragh; Silkstone, Gary; Poddar, Soumya; Ranson, Richard; Prina-Mello, Adriele; Wilson, Michael T.; Coey, J. M. D.

    2012-01-01

    The influence of isotopically enriched magnesium on the creatine kinase catalyzed phosphorylation of adenosine diphosphate is examined in two independent series of experiments where adenosine triphosphate (ATP) concentrations were determined by a luciferase-linked luminescence end-point assay or a real-time spectrophotometric assay. No increase was observed between the rates of ATP production with natural Mg, 24Mg, and 25Mg, nor was any significant magnetic field effect observed in magnetic fields from 3 to 1,000 mT. Our results are in conflict with those reported by Buchachenko et al. [J Am Chem Soc 130:12868–12869 (2008)], and they challenge these authors’ general claims that a large (two- to threefold) magnetic isotope effect is “universally observable” for ATP-producing enzymes [Her Russ Acad Sci 80:22–28 (2010)] and that “enzymatic phosphorylation is an ion-radical, electron-spin-selective process” [Proc Natl Acad Sci USA 101:10793–10796 (2005)]. PMID:22198842

  13. The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis

    PubMed Central

    Antoun, Ayman; Pavlov, Michael Y.; Andersson, Kerstin; Tenson, Tanel; Ehrenberg, Måns

    2003-01-01

    The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2. PMID:14532131

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

    PubMed Central

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

    2015-01-01

    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%. PMID:26638983

  15. Measurement and interpretation of microbial adenosine tri-phosphate (ATP) in aquatic environments.

    PubMed

    Hammes, Frederik; Goldschmidt, Felix; Vital, Marius; Wang, Yingying; Egli, Thomas

    2010-07-01

    There is a widespread need for cultivation-free methods to quantify viability of natural microbial communities in aquatic environments. Adenosine tri-phosphate (ATP) is the energy currency of all living cells, and therefore a useful indicator of viability. A luminescence-based ATP kit/protocol was optimised in order to detect ATP concentrations as low as 0.0001 nM with a standard deviation of <5%. Using this method, more than 100 water samples from a variety of aquatic environments (drinking water, groundwater, bottled water, river water, lake water and wastewater effluent) were analysed for extracellular ATP and microbial ATP in comparison with flow-cytometric (FCM) parameters. Microbial ATP concentrations ranged between 3% and 97% of total ATP concentrations, and correlated well (R(2)=0.8) with the concentrations of intact microbial cells (after staining with propidium iodide). From this correlation, we calculated an average ATP-per-cell value of 1.75x10(-10)nmol/cell. An even better correlation (R(2)=0.88) was observed between intact biovolume (derived from FCM scatter data) and microbial ATP concentrations, and an average ATP-per-biovolume value of 2.95x10(-9)nmol/microm(3) was calculated. These results support the use of ATP analysis for both routine monitoring and research purposes, and contribute towards a better interpretation of ATP data. PMID:20605621

  16. The anaerobic ribonucleoside triphosphate reductase from Escherichia coli requires S-adenosylmethionine as a cofactor.

    PubMed Central

    Eliasson, R; Fontecave, M; Jörnvall, H; Krook, M; Pontis, E; Reichard, P

    1990-01-01

    Extracts from anaerobically grown Escherichia coli contain an oxygen-sensitive activity that reduces CTP to dCTP in the presence of NADPH, dithiothreitol, Mg2+ ions, and ATP, different from the aerobic ribonucleoside diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) present in aerobically grown E. coli. After fractionation, the activity required at least five components, two heat-labile protein fractions and several low molecular weight fractions. One protein fraction, suggested to represent the actual ribonucleoside triphosphate reductase was purified extensively and on denaturing gel electrophoresis gave rise to several defined protein bands, all of which were stained by a polyclonal antibody against one of the two subunits (protein B1) of the aerobic reductase but not by monoclonal anti-B1 antibodies. Peptide mapping and sequence analyses revealed partly common structures between two types of protein bands but also suggested the presence of an additional component. Obviously, the preparations are heterogeneous and the structure of the reductase is not yet established. The second, crude protein fraction is believed to contain several ancillary enzymes required for the reaction. One of the low molecular weight components is S-adenosylmethionine; a second component is a loosely bound metal. We propose that S-adenosylmethionine together with a metal participates in the generation of the radical required for the reduction of carbon 2' of the ribosyl moiety of CTP. Images PMID:2185465

  17. Phosphatidylinositol-3,4,5-Triphosphate and Cellular Signaling: Implications for Obesity and Diabetes

    PubMed Central

    Manna, Prasenjit; Jain, Sushil K.

    2015-01-01

    Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is one of the most important phosphoinositides and is capable of activating a wide range of proteins through its interaction with their specific binding domains. Localization and activation of these effector proteins regulate a number of cellular functions, including cell survival, proliferation, cytoskeletal rearrangement, intracellular vesicle trafficking, and cell metabolism. Phosphoinositides have been investigated as an important agonist-dependent second messenger in the regulation of diverse physiological events depending upon the phosphorylation status of their inositol group. Dysregulation in formation as well as metabolism of phosphoinositides is associated with various pathophysiological disorders such as inflammation, allergy, cardiovascular diseases, cancer, and metabolic diseases. Recent studies have demonstrated that the impaired metabolism of PtdIns(3,4,5)P3 is a prime mediator of insulin resistance associated with various metabolic diseases including obesity and diabetes. This review examines the current status of the role of PtdIns(3,4,5)P3 signaling in the regulation of various cellular functions and the implications of dysregulated PtdIns(3,4,5)P3 signaling in obesity, diabetes, and their associated complications. PMID:25721445

  18. THE LOCALIZATION BY ELECTRON MICROSCOPY OF HELA CELL SURFACE ENZYMES SPLITTING ADENOSINE TRIPHOSPHATE.

    PubMed

    EPSTEIN, M A; HOLT, S J

    1963-11-01

    Cultures of normally proliferating Hela cells have been examined in thin sections by electron microscopy following glutaraldehyde fixation, staining in Wachstein and Meisel's adenosine triphosphate containing medium, postosmication, and embedding in an epoxy resin. The cells were stained in suspension in order to ensure uniform accessibility to reagents. Discrete localization of the enzyme reaction product (lead phosphate) was found at the plasma membranes of about half the cells, but nowhere else. It appeared in the form of an intensely electron-opaque deposit lying close against the outer surface of the cells and varying in amount from a chain of small particles to a dense band about 30 mmicro in width. This opaque reaction product was present over microvilli when absent elsewhere on a cell, was heaviest where microvilli and processes were profuse, and was minimal or lacking where cell surfaces were smooth. These observations are discussed in relation to both the idea that surface enzyme activity varies with the physiological phase of individual cells in a population, and the problem of how such enzyme activity becomes manifest at a given site on a morphologically changing membrane system. PMID:14086759

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

    NASA Astrophysics Data System (ADS)

    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%.

  20. Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate

    SciTech Connect

    Sharma, O.K.; Goswami, B.B.

    1981-04-01

    Extracts of interferon-treated cells synthesize unique 2',5'-linked oligo(adenylic acid) 5'-phosphates in the presence of ATP and double-stranded RNA. 2',5'-linked oligo(adenylic acid) 5'-triphosphate inhibits protein synthesis at nanomolar concentrations by activating RNase. We have observed that oligo(adenylic acid) 5'-monophosphate and 5'-triphosphate are potent inhibitors of vaccinia mRNA methylation in vitro. Both the methylation of the 5'-terminal guanine at the 7 position and the 2'-O-ribose methylation of the penultimate nucleoside are inhibited. Such inhibition of mRNA methylation is not due to degradation of the mRNA. Inhibition of the requisite modification of the 5' terminus of mRNA by 2',5'-linked oligo(adenylic acids) may be a mechanism of interferon action against both DNA and RNA viruses in which mRNAs derived from them are capped.

  1. Studies on Transcriptional Incorporation of 5’-N-Triphosphates of 5’-Amino-5’-Deoxyribonucleosides

    PubMed Central

    Kotkowiak, Weronika; Pasternak, Anna; Kierzek, Ryszard

    2016-01-01

    In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleosides (5’NH NTPs). The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5’NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5’NH NTPs incorporation. Based on experimental data it is postulated that 5’NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5’-N-triphosphates of 5’-amino-5’-deoxyribonucleoside and the exact mechanism of transcriptional inhibition. PMID:26829482

  2. Structure and Function of the E. coli Dihydroneopterin Triphosphate Pyrophosphatase: A nudix enzyme involved in Folate Biosynthesis

    SciTech Connect

    Gabelli,S.; Bianchet, M.; Lu, W.; Dunn, C.; Niu, Z.; Amzel, L.

    2007-01-01

    Nudix hydrolases are a superfamily of pyrophosphatases, most of which are involved in clearing the cell of potentially deleterious metabolites and in preventing the accumulation of metabolic intermediates. We determined that the product of the orf17 gene of Escherichia coli, a Nudix NTP hydrolase, catalyzes the hydrolytic release of pyrophosphate from dihydroneopterin triphosphate, the committed step of folate synthesis in bacteria. That this dihydroneopterin hydrolase (DHNTPase) is indeed a key enzyme in the folate pathway was confirmed in vivo: knockout of this gene in E. coli leads to a marked reduction in folate synthesis that is completely restored by a plasmid carrying the gene. We also determined the crystal structure of this enzyme using data to 1.8 {angstrom} resolution and studied the kinetics of the reaction. These results provide insight into the structural bases for catalysis and substrate specificity in this enzyme and allow the definition of the dihydroneopterin triphosphate pyrophosphatase family of Nudix enzymes.

  3. Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase.

    PubMed

    Matsuura, Mariko F; Shaw, Ryan W; Moses, Jennifer D; Kim, Hyo-Joong; Kim, Myong-Jung; Kim, Myong-Sang; Hoshika, Shuichi; Karalkar, Nilesh; Benner, Steven A

    2016-03-18

    One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides. PMID:26829203

  4. A sensitive aptasensor for colorimetric detection of adenosine triphosphate based on the protective effect of ATP-aptamer complexes on unmodified gold nanoparticles.

    PubMed

    Huo, Yuan; Qi, Liang; Lv, Xiao-Jun; Lai, Ting; Zhang, Jing; Zhang, Zhi-Qi

    2016-04-15

    Adenosine triphosphate (ATP) is the most direct source of energy in organisms. This study is the first to demonstrate that ATP-aptamer complexes provide greater protection for unmodified gold nanoparticles (AuNPs) against salt-induced aggregation than either aptamer or ATP alone. This protective effect was confirmed using transmission electron microscopy, dynamic light scattering, Zeta potential measurement, and fluorescence polarization techniques. Utilizing controlled particle aggregation/dispersion as a gauge, a sensitive and selective aptasensor for colorimetric detection of ATP was developed using ATP-binding aptamers as the identification element and unmodified AuNPs as the probe. This aptasensor exhibited a good linear relationship between the absorbance and the logarithm concentration of ATP within a 50-1000 nM range. ATP analogs such as guanosine triphosphate, uridine triphosphate and cytidine triphosphate resulted in little or no interference in the determination of ATP. PMID:26638040

  5. Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate.

    PubMed

    Appikatla, Sunita; Bessert, Denise; Lee, Icksoo; Hüttemann, Maik; Mullins, Chadwick; Somayajulu-Nitu, Mallika; Yao, Fayi; Skoff, Robert P

    2014-03-01

    Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients. PMID:24382809

  6. Role of Increased Guanosine Triphosphate Cyclohydrolase-1 Expression and Tetrahydrobiopterin Levels upon T Cell Activation

    PubMed Central

    Chen, Wei; Li, Li; Brod, Torben; Saeed, Omar; Thabet, Salim; Jansen, Thomas; Dikalov, Sergey; Weyand, Cornelia; Goronzy, Jorg; Harrison, David G.

    2011-01-01

    Tetrahydrobiopterin (BH4) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O2˙̄) rather than NO. The rate-limiting enzyme for BH4 production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH4 levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH4. Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH4 levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH4. Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH4 accumulation, reduced NO production, and increased T cell O2˙̄ production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH2 polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH4 in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH4 levels, NO production, and modulating T cell cytokine production. PMID:21343293

  7. Amperometric biosensor system for simultaneous determination of adenosine-5'-triphosphate and glucose.

    PubMed

    Kucherenko, Ivan S; Didukh, Daria Yu; Soldatkin, Oleksandr O; Soldatkin, Alexei P

    2014-06-01

    The majority of biosensors for adenosine-5'-triphosphate (ATP) determination are based on cascades of enzymatic reactions; therefore, they are sensitive to glucose or glycerol (depending on the enzymatic system) as well as to ATP. The presence of unknown concentrations of these substances in the sample greatly complicates the determination of ATP. To overcome this disadvantage of known biosensors, we developed a biosensor system consisting of two biosensors: the first one is based on glucose oxidase and is intended for measuring glucose concentration, and the second one is based on glucose oxidase and hexokinase and is sensitive toward both glucose and ATP. Using glucose concentration measured by the first biosensor, we can analyze the total response to glucose and ATP obtained by the second biosensor. Platinum disc electrodes were used as amperometric transducers. The polyphenilenediamine membrane was deposited onto the surface of platinum electrodes to avoid the response to electroactive substances. The effect of glucose concentration on biosensor determination of ATP was studied. The reproducibility of biosensor responses to glucose and ATP during a day was tested (relative standard deviation, RSD, of responses to glucose was 3-6% and to ATP was 8-12%) as well as storage stability of the biosensors (no decrease of glucose responses and 43% drop of ATP responses during 50 days). The measurements of ATP and glucose in pharmaceutical vials (including mixtures of ATP and glucose) were carried out. It was shown that the developed biosensor system can be used for simultaneous analysis of glucose and ATP concentrations in water solutions. PMID:24810180

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

    NASA Astrophysics Data System (ADS)

    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 < 1 suggests that the ATP/apatite adsorption process is mostly guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard ΔGads ° was estimated to only -4 kJ/mol, the large value of Nmax led to significantly negative effective ΔGads 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. Adenosine-5'-triphosphate release by Mannheimia haemolytica, lipopolysaccharide, and interleukin-1 stimulated bovine pulmonary epithelial cells.

    PubMed

    Craddick, Michael; Patel, Rakhi; Lower, Amanda; Highlander, Sarah; Ackermann, Mark; McClenahan, David

    2012-09-15

    Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1. PMID:22771196

  10. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats

    PubMed Central

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-01-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision. J. Comp. Neurol. 522:2928–2950, 2014. © 2014 Wiley Periodicals, Inc. PMID:24639102

  11. Adenosine triphosphate hydrolysis mechanism in kinesin studied by combined quantum-mechanical/molecular-mechanical metadynamics simulations.

    PubMed

    McGrath, Matthew J; Kuo, I-F Will; Hayashi, Shigehiko; Takada, Shoji

    2013-06-19

    Kinesin is a molecular motor that hydrolyzes adenosine triphosphate (ATP) and moves along microtubules against load. While motility and atomic structures have been well-characterized for various members of the kinesin family, not much is known about ATP hydrolysis inside the active site. Here, we study ATP hydrolysis mechanisms in the kinesin-5 protein Eg5 by using combined quantum mechanics/molecular mechanics metadynamics simulations. Approximately 200 atoms at the catalytic site are treated by a dispersion-corrected density functional and, in total, 13 metadynamics simulations are performed with their cumulative time reaching ~0.7 ns. Using the converged runs, we compute free energy surfaces and obtain a few hydrolysis pathways. The pathway with the lowest free energy barrier involves a two-water chain and is initiated by the Pγ-Oβ dissociation concerted with approach of the lytic water to PγO3-. This immediately induces a proton transfer from the lytic water to another water, which then gives a proton to the conserved Glu270. Later, the proton is transferred back from Glu270 to HPO(4)2- via another hydrogen-bonded chain. We find that the reaction is favorable when the salt bridge between Glu270 in switch II and Arg234 in switch I is transiently broken, which facilitates the ability of Glu270 to accept a proton. When ATP is placed in the ADP-bound conformation of Eg5, the ATP-Mg moiety is surrounded by many water molecules and Thr107 blocks the water chain, which together make the hydrolysis reaction less favorable. The observed two-water chain mechanisms are rather similar to those suggested in two other motors, myosin and F1-ATPase, raising the possibility of a common mechanism. PMID:23751065

  12. Adenosine 5′-Triphosphate Flux Through the North Inlet Marsh System †

    PubMed Central

    Chrzanowski, Thomas H.; Stevenson, L. Harold; Kjerfve, Bjorn

    1979-01-01

    The distribution, fluctuation, and short-term transport of total microbial biomass (measured as adenosine 5′-triphosphate [ATP]) was investigated in a large salt marsh creek. Hourly samples were collected synoptically for 25 h from 10 boats positioned across the 320-m width of the creek. Samples were collected from three depths ranging from 0.2 to 8.0 m. Hourly data obtained from each station were graphed, plotting depth against ATP. Subsequently, interpolated ATP values were generated for every one-tenth depth from the surface to the bottom with the use of an 11-point proportional divider. A total of 2,750 values were generated, and a mean value of 0.865 mg of ATP per m3 was determined. Maximum levels of ATP were found at high tide and minimal values were found at low tide. The distribution of ATP concentrations was found to be complex, with no suggestion of vertical stratification; however, horizontal divisions were apparent. ATP values corrected for direction of flow or velocity indicated two ebb-directed channels; however, when considered in total, there was a net import of ATP through the interface. The total import of ATP for this 25-h sampling period was calculated to be 3.58 kg, corresponding to a net transport of 39.8 mg of ATP per s through the cross section. Results suggest that detailed characterization of a creek transect in terms of ATP or any similar parameter requires the simultaneous measurements of both the concentration of the parameter in question and the velocity at the time and point from which the sample was taken. PMID:16345382

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

    PubMed

    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 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. PMID:24759703

  14. The inhibition of muscle contraction by adenosine 5' (beta, gamma-imido) triphosphate and by pyrophosphate.

    PubMed Central

    Pate, E; Cooke, R

    1985-01-01

    We have studied the inhibition of the contraction of glycerinated rabbit psoas muscle caused by ligands that bind to the ATPase site of myosin. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decreased the force and stiffness developed in isometric contractions and the velocity of shortening of isotonic contractions. The force exerted by isometric fibers was measured as a function of MgATP in the presence and absence of a constant concentration of the ligands. As the MgATP concentration decreased, the inhibition of tension caused by the ligand increased, reaching approximately 50% at 25 microM MgATP and either 2 mM MgPPi or 2 mM MgAMPPNP. The maximum velocity of shortening was also measured as a function of MgATP concentration in the presence of 1 and 2 mM MgPPi and 2.5 and 5 mM MgAMPPNP. Both ligands acted as pure competitive inhibitors with Ki = 3.0 mM for PPi and 5.1 mM for MgAMPPNP. These data show that both ligands are weak inhibitors of the contraction of fibers. The results provided information on the energetics of actin-myosin-ligand states that occur in the portion of the cross-bridge cycle where MgATP binds to myosin. A simple analysis of the inhibition of velocity suggests that MgAMPPNP binds to the actomyosin complex at this step of the cycle with an effective affinity constant of approximately 2 X 10(2) M-1. PMID:2990586

  15. Hydrogen sulfide decreases adenosine triphosphate levels in aortic rings and leads to vasorelaxation via metabolic inhibition

    PubMed Central

    Kiss, Levente; Deitch, Edwin A; Szabó, Csaba

    2014-01-01

    Aims Hydrogen sulfide (H2S) at low concentrations serves as a physiological endogenous vasodilator molecule, while at higher concentrations it can trigger cytotoxic effects. The aim of our study was to elucidate the potential mechanisms responsible for the effects of H2S on vascular tone. Main methods We measured the vascular tone in vitro in precontracted rat thoracic aortic rings and we have tested the effect of different oxygen levels and a variety of inhibitors affecting known vasodilatory pathways. We have also compared the vascular effect of high concentrations of H2S to those of pharmacological inhibitors of oxidative phosphorylation. Furthermore, we measured adenosine triphosphate (ATP)-levels in the same vascular tissues. Key findings We have found that in rat aortic rings: (1) H2S decreases ATP levels; (2) relaxations to H2S depend on the ambient oxygen concentration; (3) prostaglandins do not take part in the H2S induced relaxations; (4) the 3':5'-cyclic guanosine monophosphate (cGMP) – nitric oxide (NO) pathway does not have a role in the relaxations (5) the role of KATP channels is limited, while Cl−/HCO3− channels have a role in the relaxations. (6): We have observed that high concentrations of H2S relax the aortic rings in a fashion similar to sodium cyanide, and both agents reduce cellular ATP levels to a comparable degree. Significance H2S, a new gasotransmitter of emerging importance, leads to relaxation via Cl−/HCO3− channels and metabolic inhibition and the interactions of these two factors depend on the oxygen levels of the tissue. PMID:18790700

  16. Regulation of Escherichia coli aspartate transcarbamylase synthesis by guanosine tetraphosphate and pyrimidine ribonucleoside triphosphates.

    PubMed

    Turnbough, C L

    1983-02-01

    The effects of guanosine tetraphosphate (ppGpp) and pyrimidine ribonucleoside triphosphates on Escherichia coli aspartate transcarbamylase (ATCase) synthesis were examined. To determine the effect of ppGpp, a stringent (relA+) and relaxed (relA) isogenic pair of E. coli K-12 strains was starved for isoleucine, and the residual rate of synthesis of this enzyme was measured. It was necessary to starve the strains for uracil before the isoleucine limitation to maintain similar, low levels of UTP, the putative pyrimidine effector of ATCase synthesis. The isoleucine starvation of the stringent strain caused an immediate 10-fold increase in the intracellular concentration of ppGpp, which was coincident with the cessation of the synthesis of the enzyme. The elevated level of ppGpp then decayed until it reached an intracellular concentration similar to that found in unstarved cells. Enzyme synthesis resumed at this time. In the relaxed strain, the intracellular concentration of ppGpp did not increase upon isoleucine starvation and synthesis of the enzyme was not repressed. These experiments strongly indicated that ppGpp acts as a negative effector of ATCase synthesis. The repression of ATCase synthesis by ppGpp was demonstrated directly by using a Salmonella typhimurium (relA) in vitro coupled transcription-translation system with a lambda specialized transducing phage carrying the E. coli K-12 operon encoding the subunits of this enzyme (pyrBI) as a source of DNA. This in vitro system was also used to measure the effects of UTP and CTP on ATCase synthesis. Increasing the concentration of UTP in the in vitro reaction mixture resulted in strong repression of this synthesis, whereas increasing the CTP concentration did not affect synthesis significantly. Possible mechanisms for the regulation of pyr gene expression, including attenuation control, are discussed. PMID:6337130

  17. 2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure

    PubMed Central

    Moussavi-Harami, Farid; Razumova, Maria V.; Racca, Alice W.; Cheng, Yuanhua; Stempien-Otero, April; Regnier, Michael

    2014-01-01

    We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca2+ concentrations with dATP compared to ATP. This resulted in an increase in the Ca2+ sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (kTR) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development was increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models shows the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure. PMID:25498214

  18. Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation

    PubMed Central

    Tobias, Irene S.; Kaulich, Manuel; Kim, Peter K.; Simon, Nitya; Jacinto, Estela; Dowdy, Steven F.; King, Charles C.; Newton, Alexandra C.

    2016-01-01

    Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ. PMID:26635352

  19. Identification of an alternative nucleoside triphosphate: 5'-deoxyadenosylcobinamide phosphate nucleotidyltransferase in Methanobacterium thermoautotrophicum delta H.

    PubMed

    Thomas, M G; Escalante-Semerena, J C

    2000-08-01

    Computer analysis of the archaeal genome databases failed to identify orthologues of all of the bacterial cobamide biosynthetic enzymes. Of particular interest was the lack of an orthologue of the bifunctional nucleoside triphosphate (NTP):5'-deoxyadenosylcobinamide kinase/GTP:adenosylcobinamide-phosphate guanylyltransferase enzyme (CobU in Salmonella enterica). This paper reports the identification of an archaeal gene encoding a new nucleotidyltransferase, which is proposed to be the nonorthologous replacement of the S. enterica cobU gene. The gene encoding this nucleotidyltransferase was identified using comparative genome analysis of the sequenced archaeal genomes. Orthologues of the gene encoding this activity are limited at present to members of the domain Archaea. The corresponding ORF open reading frame from Methanobacterium thermoautotrophicum Delta H (MTH1152; referred to as cobY) was amplified and cloned, and the CobY protein was expressed and purified from Escherichia coli as a hexahistidine-tagged fusion protein. This enzyme had GTP:adenosylcobinamide-phosphate guanylyltransferase activity but did not have the NTP:AdoCbi kinase activity associated with the CobU enzyme of S. enterica. NTP:adenosylcobinamide kinase activity was not detected in M. thermoautotrophicum Delta H cell extract, suggesting that this organism may not have this activity. The cobY gene complemented a cobU mutant of S. enterica grown under anaerobic conditions where growth of the cell depended on de novo adenosylcobalamin biosynthesis. cobY, however, failed to restore adenosylcobalamin biosynthesis in cobU mutants grown under aerobic conditions where de novo synthesis of this coenzyme was blocked, and growth of the cell depended on the assimilation of exogenous cobinamide. These data strongly support the proposal that the relevant cobinamide intermediates during de novo adenosylcobalamin biosynthesis are adenosylcobinamide-phosphate and adenosylcobinamide-GDP, not adenosylcobinamide

  20. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs.

    PubMed

    Hughes, Scott J; Antoshchenko, Tetyana; Chen, Yun; Lu, Hua; Pizarro, Juan C; Park, Hee-Won

    2016-01-01

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the β-γ bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2'-deoxyATP structure showed the conformation of the bound

  1. Relaxation of rabbit psoas muscle fibres from rigor by photochemical generation of adenosine-5'-triphosphate.

    PubMed Central

    Goldman, Y E; Hibberd, M G; Trentham, D R

    1984-01-01

    Correlations have been made between the mechanical and biochemical descriptions of muscle relaxation. Skinned muscle fibres in the rigor state were incubated in a solution containing P3-1-(2-nitro)phenylethyladenosine-5'-triphosphate, 'caged ATP', an inert photolabile precursor of ATP, and free Ca2+ concentration less than 10(-8) M. The mechanical response of the fibre was monitored during relaxation initiated by liberating ATP with a pulse of 347 nm light from a frequency-doubled ruby laser. Tension first dropped and then rose briefly, before finally declining to the relaxed level. Stiffness, in phase with a sinusoidal length change, declined monotonically after the laser pulse. Out-of-phase stiffness increased briefly after a delay, then returned to the base line during the final relaxation. The development of the out-of-phase stiffness signal was taken as evidence that during the relaxation some cross-bridges were present with properties similar to those in an active contraction. The tension rise and slower phase of relaxation can be explained by a mechanism in which some of the cross-bridges reattach, generate force and finally detach in the absence of Ca2+ ions. In this model cross-bridge attachment is facilitated by protein co-operativity within the myofilaments. Detailed analysis of the mechanical transients makes other possible models for the initial tension rise unlikely. Stretching or releasing fibres prior to photolysis changed the time course of the early parts of the tension transient without significant effect on the later phases or on stiffness. The tension records from stretch, release and isometric trials converged to a final common time course of relaxation. Analysis of the convergence of tension records provided a means for measuring the cross-bridge detachment rate from the thin filament as a function of ATP concentration. The apparent second-order rate constant for detachment was at least 5 X 10(5) M-1 S-1 at 20-22 degrees C. The final

  2. Probing the ATP Site of GRP78 with Nucleotide Triphosphate Analogs

    PubMed Central

    Chen, Yun; Lu, Hua; Pizarro, Juan C.; Park, Hee-Won

    2016-01-01

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the β-γ bridge position to a carbon atom (AMPPCP), or the removal of the 2’-OH group (2’-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP’s binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2’-deoxyATP’s binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2’-deoxyATP structure showed the conformation of the

  3. Probing the ATP site of GRP78 with nucleotide triphosphate analogs

    DOE PAGESBeta

    Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; Lu, Hua; Pizarro, Juan C.; Park, Hee -Won

    2016-05-04

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATPmore » analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2'-deoxyATP structure showed the conformation of

  4. Plasma adenosine triphosphate and heat shock protein 72 concentrations after aerobic and eccentric exercise.

    PubMed

    Ogawa, Kishiko; Seta, Ryosuke; Shimizu, Takahiko; Shinkai, Shoji; Calderwood, Stuart K; Nakazato, Koichi; Takahashi, Kazue

    2011-01-01

    The endolysosome pathway has been proposed for secretion of heat shock protein (Hsp)72 with a regulatory role for extracellular adenosine triphosphate (ATP). Here, we tested the hypothesis that extracellular ATP mediates the increase in plasma Hsp72 after exercise. We measured plasma ATP Hsp72, cathepsin D, norepinephrine, free fatty acid, glucose, and myoglobin in 8 healthy young males (mean +/- SE: age, 22.3 +/- 0.3 years; height, 171.4 +/- 0.8 cm; weight, 68.8 +/- 3.1 kg; body mass index, 23.5 +/- 1.1 kg/cm2; VO2 max, 44.1 +/- 3.8 mL/kg/min) before and at 0, 10, 30, and 60 min after aerobic exercise (cycling) and elbow flexor eccentric exercise. Subjects cycled for 60 min at 70-75% VO2 max (mean +/- SE; 157.4 +/- 6.9 W). Eccentric strength exercise consisted of flexing the elbow joint to 90 degrees with motion speed set at 30 degrees/sec at extension and 10 degrees/sec at flexion. Subjects performed 7 sets of 10 eccentric actions with a set interval of 60 sec. The motion range of the elbow joint was 90 degrees-180 degrees. Compared with the levels of Hsp72 and ATP in plasma after bicycle exercise, those after eccentric exercise did not change. A significant group x time interaction was not observed for Hsp72 or ATP in plasma. A significant correlation was found between Hsp72 and ATP in plasma (r=0.79, P<0.05), but not between Hsp72 and norepinephrine (r=0.64, P=0.09) after bicycle exercise. A significant correlation between ATP and norepinephrine in plasma was found (r=0.89 P<0.01). We used stepwise multiple-regression analysis to determine independent predictors of exercise-induced elevation of eHsp72. Candidate predictor variables for the stepwise multiple-regression analysis were time (Pre, Post, Post10, Post30, Post60), exercise type (aerobic, eccentric), ATP, cathepsin D, norepinephrine, epinephrine, glucose, and FFA. In the regression model for Hsp72 in plasma, increased ATP and glucose were the strongest predictors of increased Hsp72 (ATP: R2=0.213, beta

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells

    PubMed Central

    Yang, Kuo H.; Kendrick, Racheal; Reddy, Y. Sunila; Kashuba, Angela D. M.; Troiani, Luigi; Bridges, Arlene S.; Fiscus, Susan A.; Forrest, Alan; Cohen, Myron S.

    2015-01-01

    The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure. PMID:26239974

  7. Cross-linked polymeric nanogel formulations of 5'-triphosphates of nucleoside analogues: role of the cellular membrane in drug release.

    PubMed

    Vinogradov, Serguei V; Kohli, Ekta; Zeman, Arin D

    2005-01-01

    Activation of cytotoxic nucleoside analogues in vivo depends primarily on their cell-specific phosphorylation. Anticancer chemotherapy using nucleoside analogues may be significantly enhanced by intracellular administration of active phosphorylated drugs. However, the cellular transport of anionic compounds is very ineffective and restricted by many drug efflux transporters. Recently developed cationic nanogel carriers can encapsulate large amounts of nucleoside 5'-triphosphates that form polyionic complexes with protonated amino groups on the polyethylenimine backbone of the nanogels. In this paper, the 5'-triphosphate of an antiviral nucleoside analogue, 3'-azido-2',3'-dideoxythymidine (AZT), was efficiently synthesized and its complexes with nanogels were obtained and evaluated as potential cytotoxic drug formulations for treatment of human breast carcinoma cells. A selective phosphorylating reagent, tris-imidazolylphosphate, was used to convert AZT into the nucleoside analogue 5'-triphosphate using a one-pot procedure. The corresponding 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) was isolated with high yield (75%). Nanogels encapsulated up to 30% of AZTTP by weight by mixing solutions of the carrier and the drug. The AZTTP/nanogel formulation showed enhanced cytotoxicity in two breast cancer cell lines, MCF-7 and MDA-MB-231, demonstrating IC50 values 130-200 times lower than those values for AZT alone. The exact mechanism of drug release from nanogels remains unclear. One mechanism could involve interaction with negatively charged counterions. A high affinity of nanogels to isolated cellular membranes has been observed, especially for nanogels made of amphiphilic block copolymer, Pluronic P85. Cellular trafficking of nanogel particles, contrasted by polyethylenimine-coordinated copper(II) ions, was studied by transmission electron microscopy (TEM), which revealed membranotropic properties of nanogels. A substantial release of encapsulated drug was

  8. Assessment of an innovative antimicrobial surface disinfectant in the operating room environment using adenosine triphosphate bioluminescence assay.

    PubMed

    Lewis, Brian D; Spencer, Maureen; Rossi, Peter J; Lee, Cheong J; Brown, Kellie R; Malinowski, Michael; Seabrook, Gary R; Edmiston, Charles E

    2015-03-01

    Terminal cleaning in the operating room is a critical step in preventing the transmission of health care-associated pathogens. The persistent disinfectant activity of a novel isopropyl alcohol/organofunctional silane solution (ISO) was evaluated in 4 operating rooms after terminal cleaning. Adenosine triphosphate bioluminescence documented a significant difference (P < .048) in surface bioburden on IOS-treated surfaces versus controls. RODAC plate cultures revealed a significant (P < .001) reduction in microbial contamination on IOS-treated surfaces compared with controls. Further studies are warranted to validate the persistent disinfectant activity of ISO within selective health care settings. PMID:25728155

  9. Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex†,‡

    PubMed Central

    Endrizzi, James A.; Kim, Hanseong; Anderson, Paul M.; Baldwin, Enoch P.

    2010-01-01

    Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447–6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface. PMID:16216072

  10. Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase.

    PubMed

    Zhang, Jiao; Qian, Yahui; Ding, Qingbao; Ou, Ling

    2015-12-28

    A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25°C for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'- triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP. PMID:26370798

  11. A review comparing deoxyribonucleoside triphosphate (dNTP) concentrations in the mitochondrial and cytoplasmic compartments of normal and transformed cells

    PubMed Central

    Gandhi, Vishal V.; Samuels, David C.

    2011-01-01

    The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10-7) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm. PMID:21774628

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    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.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. Electronic supplementary information (ESI) available: Supplementary figures related to characterization, computational studies and protein conjugation. See DOI: 10.1039/c5nr01519g

  13. The characterization of EIAV reverse transcriptase and its inhibition by 5'-triphosphates of 2'-deoxyuridine analogs, PFA and PAA.

    PubMed

    Tao, P; Zhang, X; Quan, K

    1990-01-01

    A characterization of equine infectious anemia virus reverse transcriptase (EIAV RT) and its inhibition by 5'-triphosphate analogs was undertaken to explore the possibility of using EIAV RT as an in vitro model for studying human immunodeficiency virus (HIV). EIAV RT activity was found to be dependent on the bivalent cations Mg++ and Mn++. The optimal pH for enzyme reaction was pH 8.2. EIAV RT preferred a 70 mmol/L concentration of monovalent salts. Phosphonoformic acid (PFA) was an active inhibitor of EIAV RT, but phosphonoacetic acid (PAA) and N-ethylmaleimide (NEM) were not. The inhibition of EIAV RT activity by 5'-triphosphates of nucleoside derivatives was in the following decreasing order: FLTTP greater than AZTTP greater than nPrearaUTP greater than nPredUTP = CEdUTP greater than EtdUTP greater than nPrdUTP greater than HMdUTP. nPrearaUTP was a linear competitive and PFA a linear noncompetitive inhibitor of EIAV RT with respect to dTTP. Apparent Kis and Kii were 1.5 and 2.2 mumol/L respectively. The susceptibility pattern of EIAV RT to inhibitors was similar to that of HIV RT. PMID:1711694

  14. Determination of adenosine disodium triphosphate (ATP) using oxytetracycline-Eu 3+ as a fluorescence probe by spectrofluorimetry

    NASA Astrophysics Data System (ADS)

    Hou, Faju; Miao, Yanhong; Jiang, Chongqiu

    2005-10-01

    A new spectrofluorimetric method was developed for determination of adenosine disodium triphosphate (ATP). We studied the interactions between oxytetracycline (OTC)-Eu 3+ complex and adenosine disodium triphosphate (ATP) by using UV-vis absorption and fluorescence spectra. Using oxytetracycline (OTC)-Eu 3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the OTC-Eu 3+ complex at λ = 612 nm and the enhanced fluorescence intensity of Eu 3+ ion is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP are 8.00 × 10 -8-1.50 × 10 -6 mol L -1 with detection limits of 2.67 × 10 -9 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 samples. The mechanism of fluorescence enhancement between oxytetracycline (OTC)-Eu 3+ complex and ATP was also studied.

  15. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

    1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are

  16. Inhibition of human immunodeficiency virus type 1 reverse transcriptase by the 5'-triphosphate beta enantiomers of cytidine analogs.

    PubMed Central

    Faraj, A; Agrofoglio, L A; Wakefield, J K; McPherson, S; Morrow, C D; Gosselin, G; Mathe, C; Imbach, J L; Schinazi, R F; Sommadossi, J P

    1994-01-01

    (-)-beta-L-2',3'-Dideoxycytidine (L-ddC) and (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (L-FddC) have been reported to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in vitro. In the present study, the 5'-triphosphates of L-ddC (L-ddCTP) and L-FddC (L-FddCTP) were demonstrated to competitively inhibit HIV-1 reverse transcriptase (RT), with inhibition constants (KiS) of 2 and 1.6 microM, respectively, when a poly(rI).oligo(dC)10-15 template primer was used; in comparison Ki values for beta-D-2',3'-dideoxycytidine 5'-triphosphate (D-ddCTP) and beta-D-2',3'-dideoxy-5-fluorocytidine 5'-triphosphate (D-FddCTP) were 1.1 and 1.4 microM, respectively. Use of the mutant RT at position 184 (substitution of methionine to valine [M184V]), which is associated with resistance to beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), resulted in significant increases (50- to 60-fold) in Ki values for L-ddCTP and L-FddCTP, whereas the elevation in Ki values for D-ddCTP and D-FddCTP was moderate (2-fold). L-ddCTP and L-FddCTP did not inhibit human DNA polymerases alpha and beta up to 100 microM. In contrast, D-ddCTP and D-FddCTP inhibited human DNA polymerase beta, with Ki values of 0.5 and 2.5 microM, respectively. By using sequencing analysis, L-ddCTP and L-FddCTP exhibited DNA chain-terminating activities toward the parental HIV-1 RT, whereas they were not a substrate for the mutant M184V HIV-1 RT.L-ddC and L-FddC did not inhibit the mitochondrial DNA content of human cells up to a concentration of 10 microM, whereas D-ddC and D-FddC decreased the mitochondrial DNA content by 90% at concentrations of 1 and 10 microM, respectively. All of these results suggest that further development of L-ddC, and L-FddC in particular, is warranted as a possible anti-HIV candidate. Images PMID:7530932

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

    PubMed

    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. PMID:27221644

  18. Anti-angiogenic efficacy of 5'-triphosphate siRNA combining VEGF silencing and RIG-I activation in NSCLCs.

    PubMed

    Yuan, Dongmei; Xia, Mao; Meng, Gang; Xu, Chun; Song, Yong; Wei, Jiwu

    2015-10-01

    Short interfering RNA (siRNA) targeting angiogenic factors and further inhibiting tumor angiogenesis, is one of the potent antitumor candidates for lung cancer treatment. However, this strategy must be combined with other therapeutics like chemotherapy. In this study, we designed a 5'-triphosphate siRNA targeting VEGF (ppp-VEGF), and showed that ppp-VEGF exerted three distinct antitumor effects: i) inhibition of tumor angiogenesis by silencing VEGF, ii) induction of innate immune responses by activating RIG-I signaling pathway, and thus activate antitumor immunity, iii) induction of apoptosis. In a subcutaneous model of murine lung cancer, ppp-VEGF displayed a potent antitumor effect. Our results provide a multifunctional antitumor molecule that may overcome the shortages of traditional antiangiogenic agents. PMID:26336994

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

    NASA Technical Reports Server (NTRS)

    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.

  20. Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus

    PubMed Central

    Evans, Steven J.; Fogg, Mark J.; Mamone, Anthony; Davis, Maria; Pearl, Laurence H.; Connolly, Bernard A.

    2000-01-01

    Polymerases from the Pol-I family which are able to efficiently use ddNTPs have demonstrated a much improved performance when used to sequence DNA. A number of mutations have been made to the gene coding for the Pol-II family DNA polymerase from the archaeon Pyrococcus furiosus with the aim of improving ddNTP utilisation. ‘Rational’ alterations to amino acids likely to be near the dNTP binding site (based on sequence homologies and structural information) did not yield the desired level of selectivity for ddNTPs. However, alteration at four positions (Q472, A486, L490 and Y497) gave rise to variants which incorporated ddNTPs better than the wild type, allowing sequencing reactions to be carried out at lowered ddNTP:dNTP ratios. Wild-type Pfu–Pol required a ddNTP:dNTP ratio of 30:1; values of 5:1 (Q472H), 1:3 (L490W), 1:5 (A486Y) and 5:1 (Y497A) were found with the four mutants; A486Y representing a 150-fold improvement over the wild type. A486, L490 and Y497 are on an α-helix that lines the dNTP binding groove, but the side chains of the three amino acids point away from this groove; Q472 is in a loop that connects this α-helix to a second long helix. None of the four amino acids can contact the dNTP directly. Therefore, the increased selectivity for ddNTPs is likely to arise from two factors: (i) small overall changes in conformation that subtly alter the nucleotide triphosphate binding site such that ddNTPs become favoured; (ii) interference with a conformational change that may be critical both for the polymerisation step and discrimination between different nucleotide triphosphates. PMID:10666444

  1. A new strategy for the detection of adenosine triphosphate by aptamer/quantum dot biosensor based on chemiluminescence resonance energy transfer.

    PubMed

    Zhou, Zi-Ming; Yu, Yong; Zhao, Yuan-Di

    2012-09-21

    We designed an aptasensor for the detection of adenosine triphosphate (ATP) based on chemiluminescence resonance energy transfer (CRET). An adenosine aptamer was cut into two pieces of ssDNA, which were attached to quantum dots (QDs) and horse radish peroxidase (HRP), respectively. They could reassemble into specific structures in the presence of ATP and then decrease the distance of HRP and QDs. ATP detection can be easily realized according to the fluorescent intensity of QDs, which is excited by CRET between luminol and QDs. Results show that the concentration of ATP is linear relation with the fluorescent intensity of the peak of QDs emission and the linear range for the linear equation is from 50 μM to 231 μM and the detection limit was 185 nM. When the concentration of ATP was 2 mM, the efficiency of CRET is 13.6%. Good specificity for ATP had been demonstrated compared to thymidine triphosphate (TTP), cytidine triphosphate (CTP) and guanosine triphosphate (GTP), when 1 mM of each was added, respectively. This method needs no external light source and can avoid autofluorescence and photobleaching, and ATP can be detected selectively, specifically, and sensitively in a low micromolar range, which means that the strategy reported here can be applicable to the detection of several other target molecules. PMID:22832507

  2. Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

    PubMed

    Proffitt, R T; Sankaran, L

    1976-06-29

    Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause

  3. Insights into the molecular mechanism of inhibition and drug resistance for HIV-1 RT with carbovir triphosphate.

    PubMed

    Ray, Adrian S; Yang, Zhenjun; Shi, Junxing; Hobbs, Ann; Schinazi, Raymond F; Chu, Chung K; Anderson, Karen S

    2002-04-23

    Abacavir (1592U89, or Ziagen) is a powerful and selective inhibitor of HIV-1 viral replication that has been approved by the FDA for treatment of acquired immunodeficiency syndrome. Abacavir is metabolized to the active compound carbovir triphosphate (CBVTP). This compound is a guanosine analogue containing a 2',3'-unsaturation in its planar carbocyclic deoxyribose ring that acts on HIV-1 reverse transcriptase (RT(WT)) as a molecular target, resulting in chain termination of DNA synthesis. A single amino acid change from methionine 184 to valine in HIV-1 RT (RT(M184V)) has been observed clinically in response to abacavir treatment. The ability of the natural substrate, dGTP, or CBVTP to be utilized during DNA- and RNA-directed polymerization by RT(WT) and RT(M184V) was defined by pre-steady-state kinetic parameters. In the case of RT(WT), CBVTP was found to be a surprisingly poor substrate relative to dGTP. In both DNA- and RNA-directed polymerization, a decrease in the efficiency of CBVTP utilization with respect to dGTP was found with RT(M184V), suggesting that this mutation confers resistance at the level of CBVMP incorporation. The relatively low incorporation efficiency for RT(WT) was unanticipated considering earlier studies showing that the triphosphate form of a thymidine nucleoside analogue containing a planar 2',3'-unsaturated ribose ring, D4TTP, was incorporated with high efficiency relative to the natural substrate, dTTP. The difference may be related to the isosteric replacement of oxygen in the deoxyribose ring with carbon. This hypothesis was tested by synthesizing and evaluating D4GTP (the planar 2',3'-unsaturated deoxyribose guanosine analogue that is complementary to D4TTP). In contrast to CBVTP, D4GTP was found to be an excellent substrate for RT(WT) and no resistance was conferred by the M184V mutation, thus providing novel insight into structure-activity relationships for nucleoside-based inhibitors. In this work, we illustrate how an

  4. Evidence for a "metabolically inactive" inorganic phosphate pool in adenosine triphosphate synthase reaction using localized 31P saturation transfer magnetic resonance spectroscopy in the rat brain at 11.7 T.

    PubMed

    Tiret, Brice; Brouillet, Emmanuel; Valette, Julien

    2016-09-01

    With the increased spectral resolution made possible at high fields, a second, smaller inorganic phosphate resonance can be resolved on (31)P magnetic resonance spectra in the rat brain. Saturation transfer was used to estimate de novo adenosine triphosphate synthesis reaction rate. While the main inorganic phosphate pool is used by adenosine triphosphate synthase, the second pool is inactive for this reaction. Accounting for this new pool may not only help us understand (31)P magnetic resonance spectroscopy metabolic profiles better but also better quantify adenosine triphosphate synthesis. PMID:27354096

  5. Selective and sensitive turn-on detection of adenosine triphosphate and thrombin based on bifunctional fluorescent oligonucleotide probe.

    PubMed

    Li, Feng; Du, Zongfeng; Yang, Limin; Tang, Bo

    2013-03-15

    A bifunctional fluorescent oligonucleotide probe for small molecules and protein detection has been developed based on turn on fluorescence response via the target induced structure-switching of molecular beacon. The two loops of this molecular beacon are designed in such a manner that they consist of thrombin (Tmb) aptamer sequence and adenosine triphosphate (ATP) aptamer sequence, respectively, which are utilized to sense thrombin and ATP. The oligonucleotide forms double stem-loops in the absence of targets, yielding no fluorescence emission because of the FRET from the excited fluorophore to the proximal quencher. Upon addition of the target, the ATP or Tmb, its specific interaction with loop sequence of the hairpin structure induce the separation of reporter fluorophore and the fluorescence quencher of the molecular beacon, resulting in an increase of fluorescence response. Hence, the separate analysis of ATP and Tmb could be realized through only one designed molecular beacon. The detection limits were estimated to be 25 nM for ATP and 12 nM for Tmb, respectively. The results of this study should substantially broaden the perspective for future development of oligonucleotide probe for analysis of other analytes. PMID:23102434

  6. SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.

    PubMed

    Lahouassa, Hichem; Daddacha, Waaqo; Hofmann, Henning; Ayinde, Diana; Logue, Eric C; Dragin, Loïc; Bloch, Nicolin; Maudet, Claire; Bertrand, Matthieu; Gramberg, Thomas; Pancino, Gianfranco; Priet, Stéphane; Canard, Bruno; Laguette, Nadine; Benkirane, Monsef; Transy, Catherine; Landau, Nathaniel R; Kim, Baek; Margottin-Goguet, Florence

    2012-03-01

    SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate. PMID:22327569

  7. Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme.

    PubMed

    Khrenova, Maria G; Grigorenko, Bella L; Kolomeisky, Anatoly B; Nemukhin, Alexander V

    2015-10-01

    Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H2O → Ras·GAP·GDP·Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates. PMID:26374425

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

    PubMed

    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. PMID:25683636

  9. Peroxynitrite-Dependent Zinc Release and Inactivation of Guanosine 5′-Triphosphate Cyclohydrolase 1 Instigate Its Ubiquitination in Diabetes

    PubMed Central

    Zhao, Yu; Wu, Jiliang; Zhu, Huaiping; Song, Ping; Zou, Ming-Hui

    2013-01-01

    Aberrant degradation of guanosine 5′-triphosphate cyclohydrolase 1 (GTPCH1) with consequent deficiency of tetrahydrobiopterin is considered the primary cause for endothelial dysfunction in diabetes. How GTPCH1 becomes susceptible to the degradation remains unknown. We hypothesized that oxidation and release of the zinc ion by peroxynitrite (ONOO−), a potent oxidant generated by nitric oxide and superoxide anions, instigates GTPCH1 ubiquitination and degradation. Zinc contents, GTPCH1 ubiquitination, and GTPCH1 activity were assayed in purified GTPCH1, endothelial cells, and hearts from diabetic mice. Exogenous ONOO− dose-dependently released zinc, inhibited its activity, and increased the ubiquitin binding affinity of GTPCH1 in vitro and in endothelial cells. Consistently, high glucose (30 mmol/L) inhibited GTPCH1 activity with increased ubiquitination, which was inhibited by antioxidants. Furthermore, mutation of the zinc-binding cysteine (141) (C141R or C141A) significantly reduced GTPCH1 activity and reduced its half-life but increased GTPCH1 ubiquitination, indicating an essential role of the zinc ion in maintaining the catalytic activity and stability of GTPCH1. Finally, GTPCH1 ubiquitination and degradation markedly increased in parallel with decreased GTPCH1 activity in the aortas and hearts of diabetic mice, both of which were attenuated by the inhibitors of ONOO− in mice in vivo. Taken together, we conclude that ONOO− releases zinc and inhibits GTPCH1, resulting in its ubiquitination and degradation of the enzyme. PMID:23974923

  10. Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail.

    PubMed

    Mittelstädt, Gerd; Moggré, Gert-Jan; Panjikar, Santosh; Nazmi, Ali Reza; Parker, Emily J

    2016-08-01

    Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. PMID:27191057

  11. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2.

    PubMed

    Peng, Shuang; Gerasimenko, Julia V; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Petersen, Ole H; Gerasimenko, Oleg V

    2016-08-01

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca(2+) signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca(2+) elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca(2+) signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5-10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca(2+) release followed by Ca(2+) entry and also substantially reduced Ca(2+) extrusion because of decreased intracellular ATP levels. The toxic Ca(2+) signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca(2+) signals and necrosis. We tested the effects of inhibiting the Ca(2+) release-activated Ca(2+) entry by the Ca(2+) channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca(2+) entry and also protected effectively against the development of necrosis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. PMID:27377732

  12. Measurement of Intracellular Ribavirin Mono-, Di- and Triphosphate Using Solid Phase Extraction and LC-MS/MS Quantification

    PubMed Central

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

    2014-01-01

    Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-13C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection. PMID:25555148

  13. Directional control of WAVE2 membrane targeting by EB1 and phosphatidylinositol 3,4,5-triphosphate.

    PubMed

    Takahashi, Kazuhide; Tanaka, Tacu; Suzuki, Katsuo

    2010-03-01

    Membrane targeting of WAVE2 along microtubules is mediated by a motor protein kinesin and requires Pak1, a downstream effector of Rac1. However, the mechanism by which WAVE2 targeting to the leading edge is directionally controlled remains largely unknown. Here we demonstrate that EB1, a microtubule plus-end-binding protein, constitutively associates with stathmin, a microtubule-destabilizing protein, in human breast cancer cells. Stimulation of the cells with insulin-like growth factor I (IGF-I) induced Pak1-dependent binding of the EB1-stathmin complex to microtubules that bear WAVE2 and colocalization of the complex with WAVE2 at the leading edge. Depletion of EB1 by small interfering RNA (siRNA) abrogated the IGF-I-induced WAVE2 targeting and stathmin binding to microtubules. On the other hand, chemotaxis chamber assays indicated that the IGF-I receptor (IGF-IR) was locally activated in the region facing toward IGF-I. In addition, IGF-I caused phosphatidylinositol 3-kinase (PI 3-kinase)-dependent production of phosphatidylinositol 3,4,5-triphosphate (PIP3) near activated IGF-IR and WAVE2 colocalization with it. Collectively, WAVE2-membrane targeting is directionally controlled by binding of the EB1-stathmin complex to WAVE2-bearing microtubules and by the interaction between WAVE2 and PIP3 produced near IGF-IR that is locally activated by IGF-I. PMID:19925864

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

    PubMed Central

    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

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

    PubMed Central

    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

  16. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2

    PubMed Central

    Peng, Shuang; Gerasimenko, Julia V.; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Gerasimenko, Oleg V.

    2016-01-01

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca2+ signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca2+ elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca2+ signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5–10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca2+ release followed by Ca2+ entry and also substantially reduced Ca2+ extrusion because of decreased intracellular ATP levels. The toxic Ca2+ signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca2+ signals and necrosis. We tested the effects of inhibiting the Ca2+ release-activated Ca2+ entry by the Ca2+ channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca2+ entry and also protected effectively against the development of necrosis. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377732

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

    SciTech Connect

    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.

  18. The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine*

    PubMed Central

    Hemsworth, Glyn R.; Moroz, Olga V.; Fogg, Mark J.; Scott, Benjamin; Bosch-Navarrete, Cristina; González-Pacanowska, Dolores; Wilson, Keith S.

    2011-01-01

    Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds. PMID:21454646

  19. Influence of Thromboxane A2 on the Regulation of Adenosine Triphosphate-Sensitive Potassium Channels in Mouse Ventricular Myocytes

    PubMed Central

    Jeong, In Seok; Cho, Hwa Jin; Cho, Jeong Gwan; Kim, Sang Hyung; Na, Kook Joo

    2016-01-01

    Background and Objectives Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels play an important role in myocardial protection. We examined the effects of thromboxane A2 on the regulation of KATP channel activity in single ventricular myocytes. Subjects and Methods Single ventricular myocytes were isolated from the hearts of adult Institute of Cancer Research (ICR) mice by enzymatic digestion. Single channel activity was recorded by excised inside-out and cell-attached patch clamp configurations at −60 mV holding potential during the perfusion of an ATP-free K-5 solution. Results In the excised inside-out patches, the thromboxane A2 analog, U46619, decreased the KATP channel activity in a dose-dependent manner; however, the thromboxane A2 receptor antagonist, SQ29548, did not significantly attenuate the inhibitory effect of U46619. In the cell-attached patches, U46619 inhibited dinitrophenol (DNP)-induced KATP channel activity in a dose-dependent manner, and SQ29548 attenuated the inhibitory effects of U46619 on DNP-induced KATP channel activity. Conclusion Thromboxane A2 may inhibit KATP channel activity, and may have a harmful effect on ischemic myocardium. PMID:27482267

  20. Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

    USGS Publications Warehouse

    Bushon, R.N.; Brady, A.M.; Likirdopulos, C.A.; Cireddu, J.V.

    2009-01-01

    Aims: The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water. Methods and Results: Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 and 0.77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci. Conclusions: The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria. Significance and Impact of the Study: The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications. ?? 2009 The Authors.

  1. Light-driven artificial enzymes for selective oxidation of guanosine triphosphate using water-soluble POSS network polymers.

    PubMed

    Jeon, Jong-Hwan; Tanaka, Kazuo; Chujo, Yoshiki

    2014-09-01

    The light-driven artificial enzymes were constructed to realize unnatural reactions concerning bio-significant molecules. In this manuscript, the guanosine triphosphate (GTP)-selective oxidation is reported using the network polymers composed of polyhedral oligomeric silsesquioxane (POSS). We synthesized the water-soluble POSS network polymer containing the naphthyridine ligands to capture GTP inside the networks and the ruthenium complexes to oxidize the captured GTP under light irradiation. Initially, the binding affinities of the guanosine nucleosides to the naphthyridine ligand inside the POSS network polymer were evaluated from the emission quenching experiments. Accordingly, it was observed that the naphthyridine ligand can form the stable complex only with GTP (K(a) = 5.5 × 10(6) M(-1)). These results indicate that only GTP can be captured by the network polymer. Next, the photo-catalytic activity of the ruthenium complex-modified POSS network polymer was investigated. Finally, it was revealed that the network polymer can decompose GTP efficiently under light irradiation. This is the first example, to the best of our knowledge, to offer not only the GTP-selective host polymers but also the light-driven artificial enzyme for GTP oxidation. PMID:25026217

  2. Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells.

    PubMed

    Yoneshima, Yasuto; Abolhassani, Nona; Iyama, Teruaki; Sakumi, Kunihiko; Shiomi, Naoko; Mori, Masahiko; Shiomi, Tadahiro; Noda, Tetsuo; Tsuchimoto, Daisuke; Nakabeppu, Yusaku

    2016-01-01

    Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity. PMID:27618981

  3. Probing the Interaction at the Nano–Bio Interface Using Raman Spectroscopy: ZnO Nanoparticles and Adenosine Triphosphate Biomolecules

    PubMed Central

    2015-01-01

    With the advent of nanobiotechnology, there will be an increase in the interaction between engineered nanomaterials and biomolecules. Nanoconjugates with cells, organelles, and intracellular structures containing DNA, RNA, and proteins establish sequences of nano–bio boundaries that depend on several intricate complex biophysicochemical reactions. Given the complexity of these interactions, and their import in governing life at the molecular level, it is extremely important to begin to understand such nanoparticle–biomaterial association. Here we report a unique method of probing the kinematics between an energy biomolecule, adenosine triphosphate (ATP), and hydrothermally synthesized ZnO nanostructures using micro Raman spectroscopy, X-ray diffraction, and electron microscopy experiments. For the first time we have shown by Raman spectroscopy analysis that the ZnO nanostructures interact strongly with the nitrogen (N7) atom in the adenine ring of the ATP biomolecule. Raman spectroscopy also confirms the importance of nucleotide base NH2 group hydrogen bonding with water molecules and phosphate group ionization and their pH dependence. Calculation of molecular bond force constants from Raman spectroscopy reinforces our experimental data. These data present convincing evidence of pH-dependent interactions between ATP and zinc oxide nanomaterials. Significantly, Raman spectroscopy is able to probe such difficult to study and subtle nano–bio interactions and may be applied to elegantly elucidate the nano–bio interface more generally. PMID:25152799

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

    PubMed

    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. PMID:24983417

  5. Stimulation of beta(1----3)glucan synthetase of various fungi by nucleoside triphosphates: generalized regulatory mechanism for cell wall biosynthesis.

    PubMed Central

    Szaniszlo, P J; Kang, M S; Cabib, E

    1985-01-01

    Particulate fractions from the taxonomically diverse fungi Achlya ambisexualis, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune, and Wangiella dermatitidis were found to catalyze the time-dependent incorporation of glucose from UDP-[14C]glucose into a water-insoluble material. The reaction was stimulated by bovine serum albumin. The product was characterized as beta(1----3)glucan on the basis of its resistance to alpha- and beta-amylase and susceptibility to beta(1----3)glucanase. With the exception of the preparation from A. ambisexualis, all others were stimulated by nucleoside triphosphates and their analogs. The best activators were GTP and guanosine 5'-(gamma-thio)triphosphate. It is concluded that the stimulation by nucleotides, previously found with the glucan synthetase of Saccharomyces cerevisiae, is a regulatory mechanism that was well conserved during fungal evolution, presumably because of its importance in controlling cell wall biosynthesis and cell growth. PMID:3156122

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

    PubMed Central

    Deval, Jerome; Hong, Jin; Wang, Guangyi; Taylor, Josh; Smith, Lucas K.; Fung, Amy; Stevens, Sarah K.; Liu, Hong; Jin, Zhinan; Dyatkina, Natalia; Prhavc, Marija; Stoycheva, Antitsa D.; Serebryany, Vladimir; Liu, Jyanwei; Smith, David B.; Tam, Yuen; Zhang, Qingling; Moore, Martin L.; Fearns, Rachel; Chanda, Sushmita M.; Blatt, Lawrence M.; Symons, Julian A.; Beigelman, Leo

    2015-01-01

    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. PMID:26098424

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

    PubMed

    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 < 0.05) decrease in lactate dehydrogenase (LDH) activity (marker of cellular ATP) was observed. Taken together, the hepatotoxicity of EOMABRSL could be due to the depletion of LDH and induction of oxidative damage, which may suggest possible health hazards in subjects with occupational or environmental exposure. PMID:25645823

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

    PubMed

    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. PMID:26409025

  9. Analysis of the Endogenous Deoxynucleoside Triphosphate Pool in HIV-Positive and -Negative Individuals Receiving Tenofovir-Emtricitabine.

    PubMed

    Chen, Xinhui; Castillo-Mancilla, Jose R; Seifert, Sharon M; McAllister, Kevin B; Zheng, Jia-Hua; Bushman, Lane R; MaWhinney, Samantha; Anderson, Peter L

    2016-09-01

    Tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC), two nucleos(t)ide analogs (NA), are coformulated as an anti-HIV combination tablet for treatment and preexposure prophylaxis (PrEP). TDF/FTC may have effects on the deoxynucleoside triphosphate (dNTP) pool due to their similar structures and similar metabolic pathways. We carried out a comprehensive clinical study to characterize the effects of TDF/FTC on the endogenous dNTP pool, from baseline to 30 days of TDF/FTC therapy, in both treatment-naive HIV-positive and HIV-negative individuals. dATP, dCTP, dGTP, and TTP were quantified in peripheral blood mononuclear cells (PBMC) with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. Forty individuals (19 HIV-positive) were enrolled and underwent a baseline visit and then received TDF/FTC for at least 30 days. Longitudinal measurements were analyzed using mixed-model segmented linear regression analysis. The dNTPs were reduced by 14% to 37% relative to the baseline level within 3 days in both HIV-negative and HIV-positive individuals (P ≤ 0.003). These reductions persisted to various degrees at day 30. These findings indicate that dNTP pools are influenced by TDF/FTC therapy. This may alter cellular homeostasis and could increase the antiviral effect through a more favorable analog/dNTP ratio. Further work is needed to elucidate mechanisms, to evaluate the clinical significance of these findings, and to further probe differences between HIV-negative and HIV-positive individuals. (This study has been registered at ClinicalTrials.gov under identifier NCT01040091.). PMID:27353267

  10. Effects of adenosine, adenosine triphosphate and structural analogues on glucagon secretion from the perfused pancreas of rat in vitro.

    PubMed Central

    Chapal, J.; Loubatières-Mariani, M. M.; Roye, M.; Zerbib, A.

    1984-01-01

    The effects of adenosine, adenosine triphosphate (ATP) and structural analogues have been studied on glucagon secretion from the isolated perfused pancreas of the rat in the presence of glucose (2.8 mM). Adenosine induced a transient increase of glucagon secretion. This effect was concentration-dependent in the range of 0.165 to 165 microM. ATP also induced an increase, but the effect was no greater at 165 microM than at 16.5 microM. 2-Chloroadenosine, an analogue more resistant to metabolism or uptake systems than adenosine, was more effective. Among the three structural analogues of ATP or ADP studied, beta, gamma-methylene ATP which can be hydrolyzed into AMP and adenosine had an effect similar to adenosine or ATP at the same concentrations (1.65 and 16.5 microM); in contrast alpha, beta-methylene ATP and alpha, beta-methylene ADP (resistant to hydrolysis into AMP and adenosine) were ineffective. Theophylline (50 microM) a specific blocker of the adenosine receptor, suppressed the glucagon peak induced by adenosine, 2-chloroadenosine, ATP and beta, gamma-methylene ATP (1.65 microM). An inhibitor of 5' nucleotidase, alpha, beta-methylene ADP (16.5 microM), reduced the glucagon increase induced by ATP and did not affect the response to adenosine (1.65 microM). These results support the hypothesis of adenosine receptors (P1-purinoceptors) on the pancreatic glucagon secretory cells and indicate that ATP acts after hydrolysis to adenosine. PMID:6097328

  11. Diastolic Dysfunction Induced by a High-Fat Diet Is Associated with Mitochondrial Abnormality and Adenosine Triphosphate Levels in Rats

    PubMed Central

    Kang, Ki-Woon; Kim, Ok-Soon; Chin, Jung Yeon; Kim, Won Ho; Park, Sang Hyun; Choi, Yu Jeong; Shin, Jong Ho; Jung, Kyung Tae; Lim, Do-Seon

    2015-01-01

    Background Obesity is well-known as a risk factor for heart failure, including diastolic dysfunction. However, this mechanism in high-fat diet (HFD)-induced obese rats remain controversial. The purpose of this study was to investigate whether cardiac dysfunction develops when rats are fed with a HFD for 10 weeks; additionally, we sought to investigate the association between mitochondrial abnormalities, adenosine triphosphate (ATP) levels and cardiac dysfunction. Methods We examined myocardia in Wistar rats after 10 weeks of HFD (45 kcal% fat, n=6) or standard diet (SD, n=6). Echocardiography, histomorphologic analysis, and electron microscopy were performed. The expression levels of mitochondrial oxidative phosphorylation (OXPHOS) subunit genes, peroxisome-proliferator-activated receptor γ co-activator-1α (PGC1α) and anti-oxidant enzymes were assessed. Markers of oxidative stress damage, mitochondrial DNA copy number and myocardial ATP level were also examined. Results After 10 weeks, the body weight of the HFD group (349.6±22.7 g) was significantly higher than that of the SD group (286.8±14.9 g), and the perigonadal and epicardial fat weights of the HFD group were significantly higher than that of the SD group. Histomorphologic and electron microscopic images were similar between the two groups. However, in the myocardium of the HFD group, the expression levels of OXPHOS subunit NDUFB5 in complex I and PGC1α, and the mitochondrial DNA copy number were decreased and the oxidative stress damage marker 8-hydroxydeoxyguanosine was increased, accompanied by reduced ATP levels. Conclusion Diastolic dysfunction was accompanied by the mitochondrial abnormality and reduced ATP levels in the myocardium of 10 weeks-HFD-induced rats. PMID:26790384

  12. Inosine Triphosphate Pyrophosphohydrolase (ITPA) polymorphic sequence variants in adult hematological malignancy patients and possible association with mitochondrial DNA defects

    PubMed Central

    2013-01-01

    Background Inosine triphosphate pyrophosphohydrolase (ITPase) is a ‘house-cleaning’ enzyme that degrades non-canonical (‘rogue’) nucleotides. Complete deficiency is fatal in knockout mice, but a mutant polymorphism resulting in low enzyme activity with an accumulation of ITP and other non-canonical nucleotides, appears benign in humans. We hypothesised that reduced ITPase activity may cause acquired mitochondrial DNA (mtDNA) defects. Furthermore, we investigated whether accumulating mtDNA defects may then be a risk factor for cell transformation, in adult haematological malignancy (AHM). Methods DNA was extracted from peripheral blood and bone marrow samples. Microarray-based sequencing of mtDNA was performed on 13 AHM patients confirmed as carrying the ITPA 94C>A mutation causing low ITPase activity, and 4 AHM patients with wildtype ITPA. The frequencies of ITPA 94C>A and IVS2+21A>C polymorphisms were studied from 85 available AHM patients. Results ITPA 94C>A was associated with a significant increase in total heteroplasmic/homoplasmic mtDNA mutations (p<0.009) compared with wildtype ITPA, following exclusion of haplogroup variants. This suggested that low ITPase activity may induce mitochondrial abnormalities. Compared to the normal population, frequencies for the 94C>A and IVS2+21A>C mutant alleles among the AHM patients were higher for myelodyplastic syndrome (MDS) - but below significance; were approximately equivalent for chronic lymphoblastic leukemia; and were lower for acute myeloid leukemia. Conclusions This study invokes a new paradigm for the evolution of MDS, where nucleotide imbalances produced by defects in ‘house-cleaning’ genes may induce mitochondrial dysfunction, compromising cell integrity. It supports recent studies which point towards an important role for ITPase in cellular surveillance of rogue nucleotides. PMID:23547827

  13. The N-terminal adenosine triphosphate binding domain of Hsp90 is necessary and sufficient for interaction with estrogen receptor.

    PubMed

    Bouhouche-Chatelier, L; Chadli, A; Catelli, M G

    2001-10-01

    To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being more efficient when the charged region A was present in the mutant (1-334). The N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1-224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER. PMID:11795466

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

    SciTech Connect

    Mahmood, R.

    1985-01-01

    The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz/sub 2/ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz/sub 2/ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF/sub 1/) with a binding constant of 3.2 x 10/sup 5/ M/sup -1/. Bz/sub 2/ATP is also a substrate for the ATPase activity of SF/sub 1/ in the presence of different cations. The irradiation of SF/sub 1/ with (/sup 3/H)Bz/sub 2/ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF/sub 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 (/sup 3/H)Bz/sub 2/ATP is trapped on SF/sub 1/ by cross-linking the two reactive thiols, SH/sub 1/ and SH/sub 2/, by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified (/sup 14/C)Bz/sub 2/ATP-SF/sub 1/, after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography.

  15. Lack of correlation between Legionella colonization and microbial population quantification using heterotrophic plate count and adenosine triphosphate bioluminescence measurement.

    PubMed

    Duda, Scott; Baron, Julianne L; Wagener, Marilyn M; Vidic, Radisav D; Stout, Janet E

    2015-07-01

    This investigation compared biological quantification of potable and non-potable (cooling) water samples using pour plate heterotrophic plate count (HPC) methods and adenosine triphosphate (ATP) concentration measurement using bioluminescence. The relationship between these measurements and the presence of Legionella spp. was also examined. HPC for potable and non-potable water were cultured on R2A and PCA, respectively. Results indicated a strong correlation between HPC and ATP measurements in potable water (R = 0.90, p < 0.001). In the make-up water and two cooling towers, the correlations between ATP and HPC were much weaker but statistically significant (make-up water: R = 0.37, p = 0.005; cooling tower 1: R = 0.52, p < 0.001; cooling tower 2: R = 0.54, p < 0.001). For potable and non-potable samples, HPC exhibited higher measurement variability than ATP. However, ATP measurements showed higher microbial concentrations than HPC measurements. Following chlorination of the cooling towers, ATP measurements indicated very low bacterial concentrations (<10 colony-forming units (CFU)/mL) despite high HPC concentrations (>1000 CFU/mL) which consisted primarily of non-tuberculous mycobacteria. HPC concentrations have been suggested to be predictive of Legionella presence, although this has not been proven. Our evaluation showed that HPC or ATP demonstrated a fair predictive capacity for Legionella positivity in potable water (HPC: receiver operating characteristic (ROC) = 0.70; ATP: ROC = 0.78; p = 0.003). However, HPC or ATP correctly classified sites as positive only 64 and 62% of the time, respectively. No correlation between HPC or ATP and Legionella colonization in non-potable water samples was found (HPC: ROC = 0.28; ATP: ROC = 0.44; p = 0.193). PMID:26038316

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

    PubMed

    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

  17. Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5'-triphosphate-binding cassette transporters far beyond an efflux pump.

    PubMed

    Lloberas, N; Rama, I; Llaudó, I; Torras, J; Cerezo, G; Cassis, L; Franquesa, M; Merino, A; Benitez-Ribas, D; Cruzado, J M; Herrero-Fresneda, I; Bestard, O; Grinyó, J M

    2013-06-01

    This study examines adenosine 5'-triphosphate-binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC-transporter inhibitors. Monocyte-derived DCs were cultured in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi-drug resistance (MDR1) and multi-drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P-glycoprotein (Pgp) expression with confocal microscopy. Up-regulation of maturation markers was observed in hypoxia and LPS-DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional-like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia-DCs induced more B lymphocyte proliferation than control-iDC (56% versus 9%), while LPS-DCs induced more CD8-lymphocyte proliferation (67% versus 16%). ABC transporter-inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC50 ): P-glycoprotein inhibition using valspodar (PSC833) 5 μM, CAS 115104-28-4 (MK571) 50 μM and probenecid 2·5 μM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated-DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC

  18. The hydrolysis activity of Adenosine triphosphate in myosin: a theoretical analysis of anomeric effects and the nature of transition state

    PubMed Central

    Yang, Yang; Cui, Qiang

    2009-01-01

    Combined quantum mechanical/molecular mechanical (QM/MM) calculations with density functional theory are employed to analyze two issues related to the hydrolysis activity of Adenosine triphosphate (ATP) in myosin. First, we compare the geometrical properties and electronic structure of ATP in the open (post-rigor) and closed (pre-powerstroke) active sites of the myosin motor domain. Compared to both solution and the open active-site cases, the scissile Pγ-O3β bond of ATP in the closed active-site is shown to be substantially elongated. Natural Bond Orbital (NBO) analysis clearly shows that this structural feature is correlated with the stronger anomeric effects in the closed active site, which involve charge transfers from the lone-pairs in the non-bridging oxygen in the γ phosphate to the anti-bonding orbital of the scissile bond. However, an energetic analysis finds that the ATP molecule is not significantly destabilized by the Pγ-O3β bond elongation. Therefore, despite the notable perturbations in the geometry and electronic structure of ATP as its environment changes from solution to the hydrolysis-competent active site, ground state destabilization is unlikely to play a major role in enhancing the hydrolysis activity in myosin. Second, two-dimensional potential energy maps are used to better characterize the energetic landscape near the hydrolysis transition state. The results indicate that the transition state region is energetically flat and a range of structures representative of different mechanisms according to the classical nomenclature (e.g., “associative”, “dissociative” and “concerted”) are very close in energy. Therefore, at least in the case of ATP hydrolysis in myosin, the energetic distinction between different reaction mechanisms following the conventional nomenclature is likely small. This study highlights the importance of (i). explicitly evaluating the relevant energetic properties for determining whether a factor is essential

  19. Randomized clinical trial of adenosine 5'-triphosphate on tumor growth and survival in advanced lung cancer patients.

    PubMed

    Agteresch, Hendrik J; Burgers, Sjaak A; van der Gaast, Ate; Wilson, J H Paul; Dagnelie, Pieter C

    2003-09-01

    We recently reported that regular infusions of adenosine 5'-triphosphate (ATP) inhibited loss of body weight and quality of life in patients with non-small cell lung cancer (NSCLC). In the present paper we investigated whether ATP affects tumor growth and survival in the same group of patients. Fifty-eight NSCLC patients (stage IIIB or IV) were randomly assigned to receive either 10 i.v. 30-h ATP infusions every 2-4 weeks over a 24-week period (n = 28) or no ATP (control patients, n = 30). ATP was given for a median of 6.5 infusions. Differences in time to progression and survival between patients in both groups were tested by means of the log-rank test and Cox regression analysis. Forty-nine patients were evaluable for tumor response. None of the evaluable patients showed a complete or partial response. Median time to progression was 3.9 months [95% confidence interval (CI) = 2.3-5.5] in the ATP group compared to 3.0 months (95% CI = 2.4-3.7) in the control group (p = 0.71). Median survival was 5.6 months (95% CI = 1.1-10.1) for the ATP group and 4.7 months (95% CI = 2.6-6.8) for the control group (p = 0.68). ATP treatment was associated with a significant increase in survival in the subgroup of weight-losing patients with stage IIIB NSCLC: in this subgroup, median survival was 9.3 months (95% CI = 2.1-16.5) for ATP-treated patients versus 3.5 months (95% CI = 2.3-4.7) for control patients (between-group difference: p = 0.009). No significant effect of ATP was observed for weight-losing patients with stage IV NSCLC or for weight-stable NSCLC patients. Although ATP as a single therapy does not lead to tumor regression or increased survival in patients with advanced lung cancer, it may prolong survival in weight-losing patients with stage IIIB lung cancer. The latter finding is intriguing, but requires confirmation in larger clinical trials. PMID:14501386

  20. Plasma Tenofovir, Emtricitabine, and Rilpivirine and Intracellular Tenofovir Diphosphate and Emtricitabine Triphosphate Pharmacokinetics following Drug Intake Cessation.

    PubMed

    Dickinson, Laura; Yapa, H Manisha; Jackson, Akil; Moyle, Graeme; Else, Laura; Amara, Alieu; Khoo, Saye; Back, David; Karolia, Zeenat; Higgs, Chris; Boffito, Marta

    2015-10-01

    Pharmacokinetic (PK) data describing a prolonged time course of antiretrovirals in plasma and peripheral blood mononuclear cells (PBMCs) are important for understanding and managing late or missed doses and to assess the appropriateness of compounds for preexposure prophylaxis (PrEP). This study aimed to evaluate the PK of coformulated tenofovir disoproxil fumarate (DF), emtricitabine, and rilpivirine in plasma and of the intracellular (IC) anabolites tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP) in healthy volunteers up to 9 days after drug cessation. Individuals received daily tenofovir DF-emtricitabine-rilpivirine (245/200/25 mg) for 14 days. Drug intake was stopped, and serial sampling occurred prior to the final dose and up to 216 h (9 days) after stopping drug intake. Concentrations were quantified and PK parameters calculated. Eighteen volunteers completed the study. The terminal elimination plasma half-lives for tenofovir and emtricitabine over 216 h (geometric mean [90% confidence interval]) were higher than those seen over 0 to 24 h (for tenofovir, 31 h [27 to 40 h] versus 13.3 h [12.5 to 15.1 h]; for emtricitabine, 41 h [36 to 54 h] versus 6.4 h (5.9 to 7.6 h]). Model-predicted IC half-lives (0 to 168 h) were 116 h (TFV-DP) and 37 h (FTC-TP). The plasma rilpivirine concentration at 216 h was 4.5 ng/ml (4.2 to 6.2 ng/ml), and half-lives over 0 to 216 h and 0 to 24 h were 47 h (41 to 59 h) and 35 h (28 to 46 h), respectively. These data contribute to our understanding of drug behavior following treatment interruption; however, adherence to therapy should be promoted. Validated plasma and IC target concentrations are necessary to allow interpretation with respect to sustained virus suppression or HIV prevention. (The trial was conducted in accordance with the Declaration of Helsinki [EudraCT 2012-002781-13].). PMID:26195515

  1. Comparison of plate counts, Petrifilm, dipslides, and adenosine triphosphate bioluminescence for monitoring bacteria in cooling-tower waters.

    PubMed

    Mueller, Sherry A; Anderson, James E; Kim, Byung R; Ball, James C

    2009-04-01

    Effective bacterial control in cooling-tower systems requires accurate and timely methods to count bacteria. Plate-count methods are difficult to implement on-site, because they are time- and labor-intensive and require sterile techniques. Several field-applicable methods (dipslides, Petrifilm, and adenosine triphosphate [ATP] bioluminescence) were compared with the plate count for two sample matrices--phosphate-buffered saline solution containing a pure culture of Pseudomonas fluorescens and cooling-tower water containing an undefined mixed bacterial culture. For the pure culture, (1) counts determined on nutrient agar and plate-count agar (PCA) media and expressed as colony-forming units (CFU) per milliliter were equivalent to those on R2A medium (p = 1.0 and p = 1.0, respectively); (2) Petrifilm counts were not significantly different from R2A plate counts (p = 0.99); (3) the dipslide counts were up to 2 log units higher than R2A plate counts, but this discrepancy was not statistically significant (p = 0.06); and (4) a discernable correlation (r2 = 0.67) existed between ATP readings and plate counts. For cooling-tower water samples (n = 62), (1) bacterial counts using R2A medium were higher (but not significant; p = 0.63) than nutrient agar and significantly higher than tryptone-glucose yeast extract (TGE; p = 0.03) and PCA (p < 0.001); (2) Petrifilm counts were significantly lower than nutrient agar or R2A (p = 0.02 and p < 0.001, respectively), but not statistically different from TGE, PCA, and dipslides (p = 0.55, p = 0.69, and p = 0.91, respectively); (3) the dipslide method yielded bacteria counts 1 to 3 log units lower than nutrient agar and R2A (p < 0.001), but was not significantly different from Petrifilm (p = 0.91), PCA (p = 1.00) or TGE (p = 0.07); (4) the differences between dipslides and the other methods became greater with a 6-day incubation time; and (5) the correlation between ATP readings and plate counts varied from system to system, was poor

  2. Oral adenosine-5’-triphosphate (ATP) administration increases blood flow following exercise in animals and humans

    PubMed Central

    2014-01-01

    Introduction Extracellular adenosine triphosphate (ATP) stimulates vasodilation by binding to endothelial ATP-selective P2Y2 receptors; a phenomenon, which is posited to be accelerated during exercise. Herein, we used a rat model to examine how different dosages of acute oral ATP administration affected the femoral blood flow response prior to, during, and after an exercise bout. In addition, we performed a single dose chronic administration pilot study in resistance trained athletes. Methods Animal study: Male Wistar rats were gavage-fed the body surface area, species adjusted human equivalent dose (HED) of either 100 mg (n=4), 400 mg (n=4), 1,000 mg (n=5) or 1,600 mg (n=5) of oral ATP as a disodium salt (Peak ATP®, TSI, Missoula, MT). Rats that were not gavage-fed were used as controls (CTL, n=5). Blood flow was monitored continuously: a) 60 min prior to, b) during and c) 90 min following an electrically-evoked leg-kicking exercise. Human Study: In a pilot study, 12 college-aged resistance-trained subjects were given 400 mg of ATP (Peak ATP®, TSI, Missoula, MT) daily for 12 weeks, and prior to an acute arm exercise bout at weeks 1, 4, 8, and 12. Ultrasonography-determined volumetric blood flow and vessel dilation in the brachial artery was measured at rest, at rest 30 minutes after supplementation, and then at 0, 3, and 6 minutes after the exercise. Results Animal Study: Rats fed 1,000 mg HED demonstrated significantly greater recovery blood flow (p < 0.01) and total blood flow AUC values (p < 0.05) compared to CTL rats. Specifically, blood flow was elevated in rats fed 1,000 mg HED versus CTL rats at 20 to 90 min post exercise when examining 10-min blood flow intervals (p < 0.05). When examining within-group differences relative to baseline values, rats fed the 1,000 mg and 1,600 mg HED exhibited the most robust increases in blood flow during exercise and into the recovery period. Human study: At weeks 1, 8, and 12, ATP supplementation significantly increased

  3. LDL-cholesterol reduction in patients with hypercholesterolemia by modulation of adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase

    PubMed Central

    Filippov, Sergey; Pinkosky, Stephen L.; Newton, Roger S.

    2014-01-01

    Purpose of review To review the profile of ETC-1002, as shown in preclinical and clinical studies, including LDL-cholesterol (LDL-C)-lowering activity and beneficial effects on other cardiometabolic risk markers as they relate to the inhibition of adenosine triphosphate-citrate lyase and the activation of adenosine monophosphate-activated protein kinase. Recent findings ETC-1002 is an adenosine triphosphate-citrate lyase inhibitor/adenosine monophosphate-activated protein kinase activator currently in Phase 2b clinical development. In seven Phase 1 and Phase 2a clinical studies, ETC-1002 dosed once daily for 2–12 weeks has lowered LDL-C and reduced high-sensitivity C-reactive protein by up to 40%, with neutral to positive effects on glucose levels, blood pressure, and body weight. Importantly, use of ETC-1002 in statin-intolerant patients has shown statin-like lowering of LDL-C without the muscle pain and weakness responsible for discontinuation of statin use by many patients. ETC-1002 has also been shown to produce an incremental benefit, lowering LDL-C as an add-on therapy to a low-dose statin. In over 300 individuals in studies of up to 12 weeks, ETC-1002 has been well tolerated with no serious adverse effects. Summary Because adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase play central roles in regulating lipid and glucose metabolism, pharmacological modulation of these two enzymes could provide an important therapeutic alternative for statin-intolerant patients with hypercholesterolemia. PMID:24978142

  4. Quantitative analysis of intracellular nucleoside triphosphates and other polar metabolites using ion pair reversed-phase liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Wu, Jianmei; Zhang, Yingtao; Wiegand, Richard; Wang, Jian; Bepler, Gerold; Li, Jing

    2015-12-01

    Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA)-hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100mM, pH 7.0), the combination of HFIP (100mM) and TEA (8.6mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5nM for CTP, UTP, dATP, dCTP, and dTTP, at 1nM for ATP, and at 5nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (<15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples. PMID:26551209

  5. Kinetics of inhibition of polyphenol oxidase mediated browning in apple juice by beta-cyclodextrin and L-ascorbate-2-triphosphate.

    PubMed

    Gacche, R N; Zore, G B; Ghole, V S

    2003-02-01

    Polyphenol Oxidase (PPO) mediated browning in raw fruits and vegetables is a major cause of quality deterioration in fruits and vegetables and derived food products. Here the rate of browning reaction in apple juice treated individually and in combination (1:1) of beta-Cyclodextrin (beta-CD) and L-Ascorbate-2-triphosphate (L-AATP) is described. It was observed that the rate of quinone formation can be minimized using a combination of beta-CD and L-AATP as compared to individual treatment with these agents. Kinetic experiments revealed that both compounds are non-competitive inhibitors of PPO. PMID:12751814

  6. Methylmercury-induced changes in target organs of suckling rat pups.

    PubMed

    Abdalla, Faida Husein; Bellé, Luziane Potrich; Bitencourt, Paula Eliete Rodrigues; da Silva, José Edson; Roman, Silvana; da Rosa, Cíntia; Schetinger, Maria Rosa; Moretto, Maria Beatriz

    2012-09-01

    Methylmercury (MeHg) is an organic form of mercury with toxic effects in multiple organs. The aim of the present investigation was to evaluate the in vivo effects of MeHg (1 and 4 mg/kg) given orally for seven consecutive days on adenosine deaminase (ADA), n-acetyl-β-D-glucosaminidase (NAG) and ecto-nucleoside triphosphate phosphohydrolase (NTPDase) activities, and on lipid peroxidation in hippocampus, cerebral cortex, kidney and liver of suckling rat pups. The results showed that NAG activity and lipid peroxidation levels increased in the kidney in both treatments, whereas urinary NAG activity increased only in the 1 mg/kg treatment. Despite the fact that the lipid peroxidation increased in both cerebral cortex and hippocampus, the latter appeared to be more vulnerable to MeHg exposure as it also had an increase in ADA activity. Thus, although dietary MeHg modified renal cell function, it did not alter histological features in suckling rat pups. The results of our investigation are of significant importance because they demonstrated responses to exposition to low doses of MeHg in target organs during the development of the rat. Especially the kidney was affected by the oral exposure to MeHg, suggesting the vulnerability of this organ at this stage of development. Moreover, the urinary NAG may provide important data that could serve as basis for risk assessment purposes following MeHg exposure. PMID:21194914

  7. Fluorometric determination of 2'-beta-fluoro-2',3'-dideoxyadenosine 5'-triphosphate, the active metabolite of a new anti-human immunodeficiency virus drug, in human lymphocytes.

    PubMed

    Dai, F; Kelley, J A; Zhang, H; Malinowski, N; Kavlick, M F; Lietzau, J; Welles, L; Yarchoan, R; Ford, H

    2001-01-01

    A sensitive precolumn derivatization method has been developed to measure the 5'-triphosphate of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine), a new anti-HIV drug, in human lymphocytes by HPLC using fluorescence detection. Reaction of chloroacetaldehyde with F-ddA triphosphate in extracts from human lymphocytes produces a highly fluorescent etheno adduct. This derivative is then separated and quantitated by reverse-phase paired-ion chromatography. Degradation of natural nucleic acid ribosides, such as ATP, using periodate oxidation simplifies the chromatogram and minimizes interference with detection of the target analyte. This method, modeled using cultured MOLT-4 T-lymphocytes, achieves a linear detector response for peak area measurements over the range 2.5 to 22.5 pmol (50-450 nM using 50 microl sample). Analyte recovery is greater than 90%, and the method achieves a limit of detection and limit of quantitation of 1.4 and 2.5 pmol per HPLC injection (50 microl sample containing cellular extract from 2.5 x 10(6) cells), respectively. Application of this method to measure F-ddATP in peripheral blood mononuclear cells from HIV-infected patients treated with F-ddA at 3.2 mg/kg twice daily for 22 days shows F-ddATP levels which range from 1.5 to 3.5 pmol/10(6) cells. PMID:11141306

  8. Biological effects of exogenous adenosine 5 prime -triphosphate on cultured mammalian cells: Evidence for a receptor mechanism and its regulation by desensitization

    SciTech Connect

    Gonzalez, F.A.

    1989-01-01

    Exogenous adenosine 5{prime}-triphosphate (ATP) mobilized intracellular calcium in human carcinoma A43l cells and in Swiss 3T3 and 3T6 mouse fibroblasts by increasing inositol trisphosphate similar to well down growth factors (platelet-derived growth factor (PDGF), epidermal growth factor (EGF), bradykinin (BK), serum). Calcium mobilization was examined by video imaging of fura-2 fluorescence is single cells, following the radioactive isotope {sup 45}Ca, and monitoring the decrease in fluorescence of cells loaded with chlortetracycline. Uridine 5{prime}-triphosphate, but not other nucleotides, mimicked ATP. Single-cell analysis revealed synchronous responses in 10 sec to ATP, BK or serum, while PDGF (3T3) and EGF (A431) produced slower signals with significant cell-to-cell variation. PDGF desensitized 3T3 cells to ATP and BK added 100 sec later but ATP or BK did not desensitized to PDGF. Homologous desensitization was seen with all agonists. Heterologous desensitization was also observed in A431 cells where ATP desensitized to serum, but serum did not to ATP. ATP-stimulated calcium entry was detected after 10 sec in A431 cells, but not in Swiss 3T6 cells. Entry started before significant efflux had occurred and did not fit the capacitance model of Putney. A 2-3 hr ATP pretreatment produced a homologous desensitization state that required 20 hr to disappear, probably due to down-regulation of the putative ATP receptors.

  9. Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase.

    PubMed Central

    Fan, X T; Sherwood, J L; Haslam, R J

    1994-01-01

    Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP

  10. Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

    PubMed

    Gicquel, Thomas; Victoni, Tatiana; Fautrel, Alain; Robert, Sacha; Gleonnec, Florence; Guezingar, Marie; Couillin, Isabelle; Catros, Véronique; Boichot, Elisabeth; Lagente, Vincent

    2014-04-01

    Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1β, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1β mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1β from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury. PMID:24472059

  11. Improved ischemic island skin flap survival with continuous intraarterial infusion of adenosine triphosphate--magnesium chloride and superoxide dismutase: a rat model.

    PubMed

    Zimmerman, T J; Sasaki, G H; Khattab, S

    1987-03-01

    Neurovascular island skin flaps are still hampered by occasional necrosis of their most distal aspect. Cells subjected to prolonged hypoxic conditions become intracellularly depleted of needed metabolic substrates and eventually die. Once hypoxic conditions are improved, ischemic tissue suffers further injury from the rapid accumulation of oxygen free radicals. This study showed 53% survival of a standard random flap constructed on the inferior epigastric neurovascular bundle of a rat. Random flap survival increased to 65% after intraarterial infusion of adenosine triphosphate--magnesium chloride (ATP-MgCl2); to 75% after superoxide dismutase infusion; and to 98% after combined ATP-MgCl2 and superoxide dismutase infusion. Neither substrate appeared to act by increasing blood flow to the ischemic tissue. PMID:3296921

  12. Macroscopic and Fluorescent Discrimination of Adenosine Triphosphate via Selective Metallo-hydrogel Formation: A Visual, Practical, and Reliable Rehearsal toward Cellular Imaging.

    PubMed

    Fang, Weiwei; Liu, Cong; Yu, Fabiao; Liu, Yaoqi; Li, Zhenhua; Chen, Lingxin; Bao, Xiaoling; Tu, Tao

    2016-08-17

    With use of simple terpyridine zinc nitrate complexes, intriguing visual recognition of adenosine triphosphate (ATP) via selective coordination assembly leading to two-component metallo-hydrogel formation has been realized. With intensive fluorescent study and density functional theory calculations, it may be inferred, besides the selective metal-ligand interaction between Zn center and phosphate groups, the intramolecular π-stacking between the planar nucleobases of ATP and the metal-hybrid aromatic ring of pincer complex strongly affected the geometry of the coordinated adducts and possible molecular self-assembly process, which constitute a completely new sensing strategy in comparison with the conventional approaches. Furthermore, in light of extreme sensitivity of pincer zinc complexes toward ATP at micromolar scale (1.85 μM) and remarkable fluorescent enhancement (ca. 44-fold) upon ATP addition, the feasibility of the low cytotoxicity pincer zinc complexes in monitoring ATP in HeLa cells has been fulfilled with confocal fluorescence microscopy. PMID:27420773

  13. Theoretical vibrational spectroscopy of intermediates and the reaction mechanism of the guanosine triphosphate hydrolysis by the protein complex Ras-GAP.

    PubMed

    Khrenova, Maria G; Grigorenko, Bella L; Nemukhin, Alexander V

    2016-09-01

    The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted. PMID:27214270

  14. Theoretical vibrational spectroscopy of intermediates and the reaction mechanism of the guanosine triphosphate hydrolysis by the protein complex Ras-GAP

    NASA Astrophysics Data System (ADS)

    Khrenova, Maria G.; Grigorenko, Bella L.; Nemukhin, Alexander V.

    2016-09-01

    The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted.

  15. Effect of Ca2+ on weak cross-bridge interaction with actin in the presence of adenosine 5'-[gamma-thio]triphosphate).

    PubMed Central

    Kraft, T; Yu, L C; Kuhn, H J; Brenner, B

    1992-01-01

    In the presence of the nucleotide analog adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), effects of Ca2+ on stiffness and equatorial x-ray diffraction patterns of single skinned fibers of the rabbit psoas muscle were studied. It is shown that cross-bridges in the presence of ATP[gamma S] have properties of the weak-binding states of the ATP hydrolysis cycle. Raising the Ca2+ concentration up to pCa 4.5 has little effect on actin affinity of cross-bridges in the presence of ATP[gamma S]. However, the rate constants for cross-bridge dissociation and reassociation from and to actin are reduced by about 2 orders of magnitude. In addition, nucleotide affinity of the cross-bridge is much smaller at high Ca2+ concentrations. Implications for interpretation of fiber stiffness recorded during isotonic shortening and the rising phase of a tetanus are discussed. PMID:1454820

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

    NASA Technical Reports Server (NTRS)

    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.

  17. Modification and translocation of Rac/Rop guanosine 5'-triphosphate-binding proteins of Scoparia dulcis in response to stimulation with methyl jasmonate.

    PubMed

    Mitamura, Toshiaki; Yamamura, Yoshimi; Kurosaki, Fumiya

    2011-01-01

    Translocation of two Rac/Rop guanosine 5'-triphosphate-binding proteins from Scoparia dulcis, Sdrac-1 and Sdrac-2, was examined employing transformed belladonna which overproduces these proteins as glutathione-S-transferase-tagged forms. The transferase activities of the fused proteins in microsomal fraction of belladonna markedly increased by the incubation with methyl jasmonate either in Sdrac-1 or Sdrac-2 transformant, while low and constant activities were observed in the untreated control. Recombinant Sdrac-2 protein was found to bind to prenyl chain in the presence of cell extracts prepared from methyl jasmonate-treated S. dulcis, however, Sdrac-1 was palmitoylated by the addition of the cell extracts. These results suggest that both Sdrac-1 and Sdrac-2 translocate to plant membranes by the stimulation with methyl jasmonate, however, targeting of these proteins is triggered by the independent modification mechanisms, palmitoylation for Sdrac-1 and prenylation for Sdrac-2. PMID:21628882

  18. Cloning and characterization of Sdga gene encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex in Scoparia dulcis.

    PubMed

    Shite, Masato; Yamamura, Yoshimi; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2008-11-01

    A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level. PMID:18981590

  19. Dielectric spectra broadening as a signature for dipole-matrix interaction. III. Water in adenosine monophosphate/adenosine-5'-triphosphate solutions.

    PubMed

    Puzenko, Alexander; Levy, Evgeniya; Shendrik, Andrey; Talary, Mark S; Caduff, Andreas; Feldman, Yuri

    2012-11-21

    In this, the third part of our series on the dielectric spectrum symmetrical broadening of water, we consider the nucleotide aqueous solutions. Where in Parts I [E. Levy et al., J. Chem. Phys. 136, 114502 (2012)] and II [E. Levy et al., J. Chem. Phys. 136, 114503 (2012)], the dipole-dipole or ion-dipole interaction had a dominant feature, now the interplay between these two types of dipole-matrix interactions will be considered. We present the results of high frequency dielectric measurements of different concentrations of adenosine monophosphate/adenosine-5'-triphosphate aqueous solutions. We observed the Cole-Cole broadening of the main relaxation peak of the solvent in the solutions. Moreover, depending on the nucleotide concentration, we observed both types of dipole-matrix interaction. The 3D trajectory approach (described in detail in Part I) is applied in order to highlight the differences between the two types of interaction. PMID:23181321

  20. Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: adenosylcobalamin destruction and formation of a nucleotide-based radical.

    PubMed

    Lohman, Gregory J S; Gerfen, Gary J; Stubbe, Joanne

    2010-02-23

    Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP) in <2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C(419) (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOI: 10.1021/bi902132u ]. After 1 h, a similar experiment revealed 0.45 equiv of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation by the sugar of F(2)CTP and the second with AdoCbl destruction. To determine the fate of [1'-(3)H]F(2)CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH(4) (NaB(2)H(4)) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an oxygen. High-field ENDOR studies with [1'-(2)H]F(2)CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway. PMID:20088568

  1. An efficient signal-on aptamer-based biosensor for adenosine triphosphate detection using graphene oxide both as an electrochemical and electrochemiluminescence signal indicator.

    PubMed

    Huang, Xiang; Li, Yuqin; Zhang, Xiaoshan; Zhang, Xin; Chen, Yaowen; Gao, Wenhua

    2015-09-01

    An efficient aptasensor was developed in which graphene oxide (GO) was employed as an indicator for both electrochemical impedance spectroscopy and electrochemiluminescence (ECL) signal generation. The aptasensor was fabricated by self-assembling the ECL probe of a thiolated adenosine triphosphate binding aptamer (ABA) tagged with a Ru complex (Ru(bpy)3(2+) derivatives) onto the surface of gold nanoparticle (AuNP) modified glassy carbon electrode (GCE). ABA immobilized onto AuNP modified GCE could strongly adsorb GO due to the strong π-π interaction between ABA and graphene oxide; ECL quenching of the Ru complex then takes place because of energy transfer and electron transfer, and a large increase of the electron transfer resistance (Ret) of the electrode. While in the presence of target adenosine triphosphate (ATP), the ABA prefers to form ABA-ATP bioaffinity complexes, which have weak affinity to graphene oxide and keep the graphene oxide away from the electrode surface, thus allowing the ECL signal enhancement, and in conjunction with the decrease of the Ret. Because of the high ECL quenching efficiency, unique structure, and electronic properties of graphene oxide, the Ret and ECL intensity versus the logarithm of ATP concentration was linear in the wide range from 10 pM to 10 nM with an ultra-low detection limit of 6.7 pM to 4.8 pM, respectively. The proposed aptasensor exhibited excellent reproducibility, stability, and outstanding selectivity, and ATP could be effectively distinguished from its analogues. More significantly, this efficient ECL aptasensor strategy based on GO acting both as an electrochemical and ECL signal indicator is general and can be easily extended to other biological binding events. PMID:26191542

  2. Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region.

    PubMed Central

    Tomoo, Koji; Shen, Xu; Okabe, Koumei; Nozoe, Yoshiaki; Fukuhara, Shoichi; Morino, Shigenobu; Ishida, Toshimasa; Taniguchi, Taizo; Hasegawa, Hiroshi; Terashima, Akira; Sasaki, Masahiro; Katsuya, Yoshio; Kitamura, Kunihiro; Miyoshi, Hiroshi; Ishikawa, Masahide; Miura, Kin-ichiro

    2002-01-01

    The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation. PMID:11879179

  3. Challenges and solutions in the bioanalysis of BMS-986094 and its metabolites including a highly polar, active nucleoside triphosphate in plasma and tissues using LC-MS/MS.

    PubMed

    Liu, Ang; Lute, John; Gu, Huidong; Wang, Bonnie; Trouba, Kevin J; Arnold, Mark E; Aubry, Anne-Françoise; Wang, Jian

    2015-09-01

    BMS-986094, a nucleotide polymerase inhibitor of the hepatitis C virus, was withdrawn from clinical trials because of a serious safety issue. To investigate a potential association between drug/metabolite exposure and toxicity in evaluations conducted after the termination of the BMS-986094 development program, it was essential to determine the levels of BMS-986094 and its major metabolites INX-08032, INX-08144 and INX-09054 in circulation and the active nucleoside triphosphate INX-09114 in target and non-target tissues. However, there were many challenges in the bioanalysis of these compounds. The chromatography challenge for the extremely polar nucleoside triphosphate was solved by applying mixed-mode chromatography which combined anion exchange and reversed-phase interactions. The LC conditions provided adequate retention and good peak shape of the analyte and showed good robustness. A strategy using simultaneous extraction but separate LC analysis of the prodrug BMS-986094 and its major circulating metabolites was used to overcome a carryover issue of the hydrophobic prodrug while still achieving good chromatography of the polar metabolites. In addition, the nucleotide analytes were not stable in the presence of endogenous enzymes. Low pH and low temperature were required for blood collection and plasma sample processing. However, the use of phosphatase inhibitor and immediate homogenization and extraction were critical for the quantitative analysis of the active triphosphate, INX-09114, in tissue samples. To alleviate the bioanalytical complexity caused by multiple analytes, different matrices, and various species, a fit-for-purpose approach to assay validation was implemented based on the needs of drug safety assessment in non-clinical (GLP or non-GLP) studies. The assay for INX-08032 was fully validated in plasma of toxicology species. The lower limit of quantification was 1.00ng/mL and the linear curve range was 1.00-500.00ng/mL using a weighted (1/x(2

  4. Mercury Promotes Catecholamines Which Potentiate Mercurial Autoimmunity and Vasodilation: Implications for Inositol 1,4,5-Triphosphate 3-Kinase C Susceptibility in Kawasaki Syndrome

    PubMed Central

    Yeter, Deniz; Deth, Richard

    2013-01-01

    Previously, we reviewed biological evidence that mercury could induce autoimmunity and coronary arterial wall relaxation as observed in Kawasaki syndrome (KS) through its effects on calcium signaling, and that inositol 1,4,5-triphosphate 3-kinase C (ITPKC) susceptibility in KS would predispose patients to mercury by increasing Ca2+ release. Hg2+ sensitizes inositol 1,4,5-triphosphate (IP3) receptors at low doses, which release Ca2+ from intracellular stores in the sarcoplasmic reticulum, resulting in delayed, repetitive calcium influx. ITPKC prevents IP3 from triggering IP3 receptors to release calcium by converting IP3 to inositol 1,3,4,5-tetrakisphosphate. Defective IP3 phosphorylation resulting from reduced genetic expressions of ITPKC in KS would promote IP3, which increases Ca2+ release. Hg2+ increases catecholamine levels through the inhibition of S-adenosylmethionine and subsequently catechol-O-methyltransferase (COMT), while a single nucleotide polymorphism of the COMT gene (rs769224) was recently found to be significantly associated with the development of coronary artery lesions in KS. Accumulation of norepinephrine or epinephrine would potentiate Hg2+-induced calcium influx by increasing IP3 production and increasing the permeability of cardiac sarcolemma to Ca2+. Norepinephrine and epinephrine also promote the secretion of atrial natriuretic peptide, a potent vasodilator that suppresses the release of vasoconstrictors. Elevated catecholamine levels can induce hypertension and tachycardia, while increased arterial pressure and a rapid heart rate would promote arterial vasodilation and subsequent fatal thromboses, particularly in tandem. Genetic risk factors may explain why only a susceptible subset of children develops KS although mercury exposure from methylmercury in fish or thimerosal in pediatric vaccines is nearly ubiquitous. During the infantile acrodynia epidemic, only 1 in 500 children developed acrodynia whereas mercury exposure was very common

  5. Interaction of Beta-Hydroxy-Beta-Methylbutyrate Free Acid and Adenosine Triphosphate on Muscle Mass, Strength, and Power in Resistance Trained Individuals.

    PubMed

    Lowery, Ryan P; Joy, Jordan M; Rathmacher, John A; Baier, Shawn M; Fuller, John C; Shelley, Mack C; Jäger, Ralf; Purpura, Martin; Wilson, Stephanie M C; Wilson, Jacob M

    2016-07-01

    Lowery, RP, Joy, JM, Rathmacher, JA, Baier, SM, Fuller, JC Jr, Shelley, MC II, Jäger, R, Purpura, M, Wilson, SMC, and Wilson, JM. Interaction of beta-hydroxy-beta-methylbutyrate free acid and adenosine triphosphate on muscle mass, strength, and power in resistance trained individuals. J Strength Cond Res 30(7): 1843-1854, 2016-Adenosine-5'-triphosphate (ATP) supplementation helps maintain performance under high fatiguing contractions and with greater fatigue recovery demands also increase. Current evidence suggests that the free acid form of β-hydroxy-β-methylbutyrate (HMB-FA) acts by speeding regenerative capacity of skeletal muscle after high-intensity or prolonged exercise. Therefore, we investigated the effects of 12 weeks of HMB-FA (3 g) and ATP (400 mg) administration on lean body mass (LBM), strength, and power in trained individuals. A 3-phase double-blind, placebo-, and diet-controlled study was conducted. Phases consisted of an 8-week periodized resistance training program (phase 1), followed by a 2-week overreaching cycle (phase 2), and a 2-week taper (phase 3). Lean body mass was increased by a combination of HMB-FA/ATP by 12.7% (p < 0.001). In a similar fashion, strength gains after training were increased in HMB-FA/ATP-supplemented subjects by 23.5% (p < 0.001). Vertical jump and Wingate power were increased in the HMB-FA/ATP-supplemented group compared with the placebo-supplemented group, and the 12-week increases were 21.5 and 23.7%, respectively. During the overreaching cycle, strength and power declined in the placebo group (4.3-5.7%), whereas supplementation with HMB-FA/ATP resulted in continued strength gains (1.3%). In conclusion, HMB-FA and ATP in combination with resistance exercise training enhanced LBM, power, and strength. In addition, HMB-FA plus ATP blunted the typical response to overreaching, resulting in a further increase in strength during that period. It seems that the combination of HMB-FA/ATP could benefit those who

  6. Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut

    PubMed Central

    Burnstock, G; Campbell, G; Satchell, D; Smythe, ANNE

    1997-01-01

    Stimulation of the vagal non-adrenergic inhibitory innervation caused the release of adenosine and inosine into vascular perfusates from the stomachs of guinea-pigs and toads. Stimulation of portions of Auerbach's plexus isolated from turkey gizzard caused the release of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). ATP, added to solutions perfused through the toad stomach vasculature, was broken down to adenosine, inosine and adenine. Of a series of purine and pyrimidine derivatives tested for inhibitory activity on the guinea-pig isolated taenia coli, ATP and ADP were the most potent. ATP caused inhibition of twelve other gut preparations previously shown to contain non-adrenergic inhibitory nerves. The inhibitory action of ATP was not prevented by tetrodotoxin. Quinidine antagonized relaxations of the guinea-pig taenia coli caused by catecholamines or adrenergic nerve stimulation. Higher concentrations of quinidine antagonized relaxations caused by ATP or non-adrenergic inhibitory nerve stimulation. When tachyphylaxis to ATP had been produced in the rabbit ileum, there was a consistent depression of the responses to non-adrenergic inhibitory nerve stimulation but not of responses to adrenergic nerve stimulation. It is suggested that ATP or a related nucleotide is the transmitter substance released by the non-adrenergic inhibitory innervation of the gut. PMID:9142414

  7. Development of an immune function assay by measuring intracellular adenosine triphosphate (iATP) levels in mitogen-stimulated CD4+ T lymphocytes.

    PubMed

    Naderi, Hadi; Najafi, Alireza; Khoshroo, Mohammad; Tajik, Nader

    2016-01-01

    We developed an immune function assay for monitoring CD4+ T cells activity based on changes in intracellular adenosine triphosphate (iATP) levels after phytohemagglutinin (PHA) stimulation. Blood samples were obtained from 40 healthy subjects and 30 RTRs and incubated with 5 µg/mL of PHA for 15-18 hr at 37°C and 5% CO2. Afterward, the CD4+ T cells were separated by antibody-coated magnetic beads and lysed. Then, iATP content in unstimulated and stimulated conditions was measured by luciferin-luciferase reaction using a log-log standard curve. The iATP levels showed significant increase in CD4+ T cells in both healthy persons (mean: 550 ± 142 ng/mL vs. 109 ± 54 ng/mL) and RTRs (mean: 394 ± 160 ng/mL vs. 52 ± 37 ng/mL) after PHA stimulation (P < 0.001). However, the iATP production in RTRs was significantly lower than that in healthy individuals; both prior to and after stimulation with PHA (P < 0.001). No gender-specific difference in iATP production was observed between women and men subjects. This rapid and low-cost assay reflects the degree of immune cell function through assessment of CD4+ T cells activation. Thus, it can be used for evaluation of immune system status in immunodeficient individuals as well as in immunosuppressed transplant recipients who needs drug adjustment. PMID:27089103

  8. Adenosine Triphosphate (ATP) Inhibits Voltage-Sensitive Potassium Currents in Isolated Hensen’s Cells and Nifedipine Protects Against Noise-Induced Hearing Loss in Guinea Pigs

    PubMed Central

    Ye, Rui; Liu, Jun; Jia, Zhiying; Wang, Hongyang; Wang, YongAn; Sun, Wei; Wu, Xuan; Zhao, Zhifei; Niu, Baolong; Li, Xingqi; Dai, Guanghai; Li, Jianxiong

    2016-01-01

    Background There is increasing evidence that adenosine triphosphate (ATP), a well-known neurotransmitter and neuromodulator in the central nervous system, plays an important role as an extracellular chemical messenger in the cochlea. Material/Methods Using a whole-cell recording technique, we studied the effects of ATP on isolated Hensen’s cells, which are supporting cells in the cochlea, to determine if they are involved in the transduction of ions with hair cells. Results ATP (0.1–10 μM) reduced the potassium current (IK+) in the majority of the recorded Hensen’s cells (21 out of 25 cells). An inward current was also induced by high concentrations of ATP (100 μM to 10 mM), which was reversibly blocked by 100 μM suramin (a purinergic antagonist) and blocked by nifedipine (an L-type calcium channel blocker). After the cochleas were perfused with artificial perilymph solutions containing nifedipine and exposed to noise, the amplitude increase in the compound action potential (CAP) threshold and the reduction in cochlear microphonics was lower than when they were exposed to noise alone. Conclusions Our results suggest that ATP can block IK+ channels at a low concentration and induce an inward Ca2+ current at high concentrations, which is reversed by purinergic receptors. Nifedipine may have a partially protective effect on noise-induced hearing loss (NIHL). PMID:27292522

  9. Application of an intracellular assay for determination of tenofovir-diphosphate and emtricitabine-triphosphate from erythrocytes using dried blood spots.

    PubMed

    Zheng, Jia-Hua; Rower, Caitlin; McAllister, Kevin; Castillo-Mancilla, Jose; Klein, Brandon; Meditz, Amie; Guida, L Anthony; Kiser, Jennifer J; Bushman, Lane R; Anderson, Peter L

    2016-04-15

    This communication describes the application of an existing intracellular methodology to the quantitation of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) from erythrocytes using dried blood spots (DBS). Concentrations were determined from a 3mm DBS punch extracted into a 70:30 methanol:water solution (lysed cellular matrix). This extraction solution was then subjected to a previously validated analytical procedure for lysed cellular matrix. Experiments for DBS validation used replicate samples from study participants to demonstrate acceptable reproducibility with spot volumes ranging from 10-50 μL and punch location either from the edge or center of the spot. Analysis of paired DBS with purified red blood cells showed that a 3mm DBS punch contained an average of 11.9 million cells for the observed hematocrit range of the participants (35-50%). Numerous stability tests were completed showing that whole blood in an EDTA vacutainer could sit for 24h at room temperature prior to spotting, and DBS could remain at room temperature for up to five days including shipment at ambient using 2-days delivery. DBS stability in storage was acceptable up to 18 months at -20°C or -80°C and DBS could undergo 4 Freeze/Thaw cycles. The described method was applied to HIV prophylaxis studies, demonstrating powerful associations with HIV acquisition through its ability to discriminate gradients of adherence. PMID:26829517

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

    PubMed

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

    2015-02-01

    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 60nM. 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 determination of adenosine in urine. PMID:25604821