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Sample records for ecto-nucleoside triphosphate diphosphohydrolase

  1. Possible Effects of Microbial Ecto-Nucleoside Triphosphate Diphosphohydrolases on Host-Pathogen Interactions

    PubMed Central

    Sansom, Fiona M.; Robson, Simon C.; Hartland, Elizabeth L.

    2008-01-01

    Summary: In humans, purinergic signaling plays an important role in the modulation of immune responses through specific receptors that recognize nucleoside tri- and diphosphates as signaling molecules. Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) have important roles in the regulation of purinergic signaling by controlling levels of extracellular nucleotides. This process is key to pathophysiological protective responses such as hemostasis and inflammation. Ecto-NTPDases are found in all higher eukaryotes, and recently it has become apparent that a number of important parasitic pathogens of humans express surface-located NTPDases that have been linked to virulence. For those parasites that are purine auxotrophs, these enzymes may play an important role in purine scavenging, although they may also influence the host response to infection. Although ecto-NTPDases are rare in bacteria, expression of a secreted NTPDase in Legionella pneumophila was recently described. This ecto-enzyme enhances intracellular growth of the bacterium and potentially affects virulence. This discovery represents an important advance in the understanding of the contribution of other microbial NTPDases to host-pathogen interactions. Here we review other progress made to date in the characterization of ecto-NTPDases from microbial pathogens, how they differ from mammalian enzymes, and their association with organism viability and virulence. In addition, we postulate how ecto-NTPDases may contribute to the host-pathogen interaction by reviewing the effect of selected microbial pathogens on purinergic signaling. Finally, we raise the possibility of targeting ecto-NTPDases in the development of novel anti-infective agents based on potential structural and clear enzymatic differences from the mammalian ecto-NTPDases. PMID:19052327

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

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  3. Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system.

    PubMed

    De Giorgi, Marco; Cinti, Alessandro; Pelikant-Malecka, Iwona; Chisci, Elisa; Lavitrano, Marialuisa; Giovannoni, Roberto; Smolenski, Ryszard T

    2015-05-01

    We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.

  4. Expression of ecto-nucleoside triphosphate diphosphohydrolase3 (NTPDase3) in the female rat brain during postnatal development.

    PubMed

    Grković, Ivana; Bjelobaba, Ivana; Mitrović, Nataša; Lavrnja, Irena; Drakulić, Dunja; Martinović, Jelena; Stanojlović, Miloš; Horvat, Anica; Nedeljković, Nadežda

    2016-11-01

    Nucleoside triphosphate diphosphohydrolase3 (NTPDase3) is membrane-bound ecto-enzyme which hydrolyzes extracellular ATP, thus modulating the function of purinergic receptors and the pattern of purinergic signaling. Here we analyzed the developmental expression of NTPDase3 in female hypothalamus, cerebral cortex and hippocampal formation at different postnatal ages (PD7-PD90) by qRT-PCR and immunohistochemistry. In hypothalamus and hippocampus, a similar developmental profile was seen: NTPDase3 gene expression was stable during postnatal development and increased in adults. In the cortex, upregulation of NTPDase3 mRNA expression was seen at PD15 and further increase was evidenced in adults. Immunohistochemical analysis at PD7 revealed faint neuronal NTPDase3 localization in a dorsal hypothalamus. The immunoreactivity (ir) gradually increased in PD15 and PD20, in clusters of cells in the lateral, ventral and dorsomedial hypothalamus. Furthermore, in PD20 animals, NTPDase3-ir was detected on short fibers in the posterior hypothalamic area, while in PD30 the fibers appeared progressively longer and markedly varicose. In adults, the strongest NTPDase3-ir was observed in collections of cells in dorsomedial hypothalamic nucleus, dorsal and lateral hypothalamus and in several thalamic areas, whereas the varicose fibers traversed entire diencephalon, particularly paraventricular thalamic nucleus, ventromedial and dorsomedial hypothalamic nuclei, the arcuate nucleus and the prefornical part of the lateral hypothalamus. The presumably ascending NTPDase3-ir fibers were first observed in PD20; their density and the varicose appearance increased until the adulthood. Prominent enhancement of NTPDase3-ir in the hypothalamus coincides with age when animals acquire diurnal rhythms of sleeping and feeding, supporting the hypothesis that this enzyme may be involved in regulation of homeostatic functions.

  5. Comparative expression of p2x receptors and ecto-nucleoside triphosphate diphosphohydrolase 3 in hypocretin and sensory neurons in zebrafish.

    PubMed

    Appelbaum, Lior; Skariah, Gemini; Mourrain, Philippe; Mignot, Emmanuel

    2007-10-12

    The hypocretin/orexin (HCRT/ORX) excitatory neuropeptides are expressed in a small population of lateral hypothalamic cells in mammals and fish. In humans, loss of these cells causes the sleep disorder narcolepsy. Identification of genes expressed in HCRT-producing cells may be revealing as to the regulation of sleep and the pathophysiology of narcolepsy. In this study, in situ hybridization analyses were performed to characterize the expression pattern of receptors and enzyme, which regulate ATP-mediated transmission in hypocretin cells of zebrafish larvae. The zebrafish cDNA encoding the ecto-nucleoside triphosphate diphosphohydrolase 3 (ENTPD3/NTPDase3) was isolated. This transcript was found to be expressed in zebrafish HCRT cells as previously reported in mammals. It was also expressed in the cranial nerves (gV, gVII, gIV and gX) and in primary sensory neurons (i.e., Rohon-Beard neurons) in the spinal cord. The expression of known zebrafish p2rx purinergic receptor family members was next studied and found to overlap with the entpd3 expression pattern. Specifically, p2rx2, p2rx3.1, p2rx3.2 and p2rx8 were expressed in the trigeminal ganglia and subsets of Rohon-Beard neurons. In contrast to mammals, p2rx2 was not expressed in HCRT cells; rather, p2rx8 was expressed with entpd3 in this hypothalamic region. The conservation of expression of these genes in HCRT cells and sensory neurons across vertebrates suggests an important role for ATP mediated transmission in the regulation of sleep and the processing of sensory inputs.

  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.

  7. Therapeutic potentials of ecto-nucleoside triphosphate diphosphohydrolase, ecto-nucleotide pyrophosphatase/phosphodiesterase, ecto-5'-nucleotidase, and alkaline phosphatase inhibitors.

    PubMed

    al-Rashida, Mariya; Iqbal, Jamshed

    2014-07-01

    The modulatory role of extracellular nucleotides and adenosine in relevance to purinergic cell signaling mechanisms has long been known and is an object of much research worldwide. These extracellular nucleotides are released by a variety of cell types either innately or as a response to patho-physiological stress or injury. A variety of surface-located ecto-nucleotidases (of four major types; nucleoside triphosphate diphosphohydrolases or NTPDases, nucleotide pyrophosphatase/phosphodiesterases or NPPs, alkaline phosphatases APs or ALPs, and ecto-5'-nucleotidase or e5NT) are responsible for meticulously controlling the availability of these important signaling molecules (at their respective receptors) in extracellular environment and are therefore crucial for maintaining the integrity of normal cell functioning. Overexpression of many of these ubiquitous ecto-enzymes has been implicated in a variety of disorders including cell adhesion, activation, proliferation, apoptosis, and degenerative neurological and immunological responses. Selective inhibition of these ecto-enzymes is an area that is currently being explored with great interest and hopes remain high that development of selective ecto-nucleotidase inhibitors will prove to have many beneficial therapeutic implications. The aim of this review is to emphasize and focus on recent developments made in the field of inhibitors of ecto-nucleotidases and to highlight their structure activity relationships wherever possible. Most recent and significant advances in field of NTPDase, NPP, AP, and e5NT inhibitors is being discussed in detail in anticipation of providing prolific leads and relevant background for research groups interested in synthesis of selective ecto-nucleotidase inhibitors.

  8. Selective Nucleoside Triphosphate Diphosphohydrolase-2 (NTPDase2) Inhibitors: Nucleotide Mimetics Derived from Uridine-5′-carboxamide†

    PubMed Central

    Brunschweiger, Andreas; Iqbal, Jamshed; Umbach, Frank; Scheiff, Anja B.; Munkonda, Mercedes N.; Sévigny, Jean; Knowles, Aileen F.; Müller, Christa E

    2016-01-01

    Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases) dephosphorylate nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present study we synthesized adenine and uracil nucleotide mimetics, in which the phosphate residues were replaced by phosphonic acid esters attached to the nucleoside at the 5′-position by amide linkers. Among the synthesized uridine derivatives, we identified the first potent and selective inhibitors of human NTPDase2. The most potent compound was 19a (PSB-6426), which was a competitive inhibitor of NTPDase2 exhibiting a Ki value of 8.2 μM and selectivity versus other NTPDases. It was inactive toward uracil nucleotide-activated P2Y2, P2Y4, and P2Y6 receptor subtypes. Compound 19a was chemically and metabolically highly stable. In contrast to the few known (unselective) NTPDase inhibitors, 19a is an uncharged molecule and may be perorally bioavailable. NTPDase2 inhibitors have potential as novel cardioprotective drugs for the treatment of stroke and for cancer therapy. PMID:18630897

  9. Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

    PubMed

    Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

    2016-01-01

    Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis.

  10. 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.

  11. An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.

    PubMed

    Maia, Ana Carolina Ribeiro Gomes; Porcino, Gabriane Nascimento; Detoni, Michelle de Lima; Emídio, Nayara Braga; Marconato, Danielle Gomes; Faria-Pinto, Priscila; Fessel, Melissa Regina; Reis, Alexandre Barbosa; Juliano, Luiz; Juliano, Maria Aparecida; Marques, Marcos José; Vasconcelos, Eveline Gomes

    2013-02-01

    We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

  12. Upregulation of nucleoside triphosphate diphosphohydrolase-1 and ecto-5'-nucleotidase in rat hippocampus after repeated low-dose dexamethasone administration.

    PubMed

    Drakulić, Dunja; Stanojlović, Miloš; Nedeljković, Nadežda; Grković, Ivana; Veličković, Nataša; Guševac, Ivana; Mitrović, Nataša; Buzadžić, Ivana; Horvat, Anica

    2015-04-01

    Although dexamethasone (DEX), a synthetic glucocorticoid receptor (GR) analog with profound effects on energy metabolism, immune system, and hypothalamic-pituitary-adrenal axis, is widely used therapeutically, its impact on the brain is poorly understood. The aim of the present study was to explore the effect of repeated low-dose DEX administration on the activity and expression of the ectonucleotidase enzymes which hydrolyze and therefore control extracellular ATP and adenosine concentrations in the synaptic cleft. Ectonucleotidases tested were ectonucleoside triphosphate diphosphohydrolase 1-3 (NTPDase1-3) and ecto-5'-nucleotidase (eN), whereas the effects were evaluated in two brain areas that show different sensitivity to glucocorticoid action, hippocampus, and cerebral cortex. In the hippocampus, but not in cerebral cortex, modest level of neurodegenerative changes as well as increase in ATP, ADP, and AMP hydrolysis and upregulation of NTPDase1 and eN mRNA expression ensued under the influence of DEX. The observed pattern of ectonucleotidase activation, which creates tissue volume with enhanced capacity for adenosine formation, is the hallmark of the response after different insults to the brain.

  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. Removal from the membrane affects the interaction of rat osseous plate ecto-nucleosidetriphosphate diphosphohydrolase-1 with substrates and ions.

    PubMed

    Garçon, Daniela P; Masui, Douglas C; Furriel, Rosa P M; Leone, Francisco A

    2008-01-01

    We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M (r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always <2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

  15. Identification of ATP diphosphohydrolase activity in human term placenta using a novel assay for AMP.

    PubMed

    Papamarcaki, T; Tsolas, O

    1990-09-03

    Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN3 were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase. A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05-0.8 microM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates. The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.

  16. 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.

  17. 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.

  18. Plasmodium falciparum GFP-E-NTPDase expression at the intraerythrocytic stages and its inhibition blocks the development of the human malaria parasite.

    PubMed

    Borges-Pereira, Lucas; Meissner, Kamila Anna; Wrenger, Carsten; Garcia, Célia R S

    2017-03-11

    Plasmodium falciparum is the causative agent of the most dangerous form of malaria in humans. It has been reported that the P. falciparum genome encodes for a single ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), an enzyme that hydrolyzes extracellular tri- and di-phosphate nucleotides. The E-NTPDases are known for participating in invasion and as a virulence factor in many pathogenic protozoa. Despite its presence in the parasite genome, currently, no information exists about the activity of this predicted protein. Here, we show for the first time that P. falciparum E-NTPDase is relevant for parasite lifecycle as inhibition of this enzyme impairs the development of P. falciparum within red blood cells (RBCs). ATPase activity could be detected in rings, trophozoites, and schizonts, as well as qRT-PCR, confirming that E-NTPDase is expressed throughout the intraerythrocytic cycle. In addition, transfection of a construct which expresses approximately the first 500 bp of an E-NTPDase-GFP chimera shows that E-NTPDase co-localizes with the endoplasmic reticulum (ER) in the early stages and with the digestive vacuole (DV) in the late stages of P. falciparum intraerythrocytic cycle.

  19. Mesenchymal stem cells from different murine tissues have differential capacity to metabolize extracellular nucleotides.

    PubMed

    Iser, Isabele C; Bracco, Paula A; Gonçalves, Carlos E I; Zanin, Rafael F; Nardi, Nance B; Lenz, Guido; Battastini, Ana Maria O; Wink, Márcia R

    2014-10-01

    Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically.

  20. 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.

  1. Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase.

    PubMed

    Murphy-Piedmonte, Deirdre M; Crawford, Patrick A; Kirley, Terence L

    2005-03-14

    The ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are a family of enzymes that control the levels of extracellular nucleotides, thereby modulating purinergically controlled physiological processes. Six of the eight known NTPDases are membrane-bound enzymes; only NTPDase 5 and 6 can be released as soluble enzymes. Here we report the first bacterial expression and refolding of soluble human NTPDase5 from inclusion bodies. The results show that NTPDase5 requires the presence of divalent cations (Mg2+ or Ca2+) for activity. Positive cooperativity with respect to hydrolysis of its preferred substrates (GDP, IDP and UDP) is observed, and this positive cooperativity is attenuated in the presence of nucleoside monophosphate products (e.g., GMP and AMP). In addition, comparing the biochemical properties of wild-type NTPDase5 and those of a mutant NTPDase5 (C15S, which lacks the single, non-conserved cysteine residue), also expressed in bacteria, suggests that Cys15 is not essential for either proper refolding or enzymatic activity (indicating this residue is not involved in a disulfide bond). Moreover, the substrate profile of bacterially expressed NTPDase5, as well as the C15S mutant, was determined to be similar to that of full-length, membrane-bound and soluble NTPDase5 expressed in mammalian COS cells.

  2. 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

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

    PubMed

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

    2016-06-01

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

  4. 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... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  5. 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... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  6. 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... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

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

    PubMed

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

    2017-03-01

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

  9. Ecto-nucleotide pyrophosphatase/phosphodiesterase as part of a multiple system for nucleotide hydrolysis by platelets from rats: kinetic characterization and biochemical properties.

    PubMed

    Fürstenau, Cristina Ribas; Trentin, Danielle Da Silva; Barreto-Chaves, Maria Luiza Morais; Sarkis, João José Freitas

    2006-03-01

    In this study, we describe an ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activity in rat platelets. Using p-nitrophenyl 5'-thymidine monophosphate (p-Nph-5'-TMP) as a substrate for E-NPP, we demonstrate an enzyme activity that shares the major biochemical properties described for E-NPPs: alkaline pH dependence, divalent cation dependence and blockade of activity by metal ion chelator. K(m) and V(max) values for p-Nph-5'-TMP hydrolysis were found to be 106 +/- 18 microM and 3.44 +/- 0.18 nmol p-nitrophenol/min/mg (mean +/- SD, n = 5). We hypothesize that an E-NPP is co-localized with an ecto-nucleoside triphosphate diphosphohydrolase and an ecto-5'-nucleotidase on the platelet surface, as part of a multiple system for nucleotide hydrolysis, since they can act under distinct physiological conditions and can be differently regulated. Thus, 0.25 mM suramin inhibited p-Nph-5'-TMP, ATP and ADP hydrolysis, while 0.5 mM AMP decreased only p-Nph-5'-TMP hydrolysis. Besides, 5.0, 10 and 20 mM sodium azide just inhibited ATP and ADP hydrolysis. Angiotensin II (5.0 and 10 nM) affected only ADP hydrolysis. Gadolinium chloride (0.2 and 0.5 mM) strongly inhibited the ATP and ADP hydrolysis. The E-NPP described here represents a novel insight into the control of platelet purinergic signaling.

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

    PubMed

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

    2015-12-01

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

  11. 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.

  12. The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates.

    PubMed

    Santner, Tobias; Siegmund, Vanessa; Marx, Andreas; Micura, Ronald

    2012-04-01

    Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.

  13. Extracellular adenosine triphosphate and adenosine in cancer.

    PubMed

    Stagg, J; Smyth, M J

    2010-09-30

    Adenosine triphosphate (ATP) is actively released in the extracellular environment in response to tissue damage and cellular stress. Through the activation of P2X and P2Y receptors, extracellular ATP enhances tissue repair, promotes the recruitment of immune phagocytes and dendritic cells, and acts as a co-activator of NLR family, pyrin domain-containing 3 (NLRP3) inflammasomes. The conversion of extracellular ATP to adenosine, in contrast, essentially through the enzymatic activity of the ecto-nucleotidases CD39 and CD73, acts as a negative-feedback mechanism to prevent excessive immune responses. Here we review the effects of extracellular ATP and adenosine on tumorigenesis. First, we summarize the functions of extracellular ATP and adenosine in the context of tumor immunity. Second, we present an overview of the immunosuppressive and pro-angiogenic effects of extracellular adenosine. Third, we present experimental evidence that extracellular ATP and adenosine receptors are expressed by tumor cells and enhance tumor growth. Finally, we discuss recent studies, including our own work, which suggest that therapeutic approaches that promote ATP-mediated activation of inflammasomes, or inhibit the accumulation of tumor-derived extracellular adenosine, may constitute effective new means to induce anticancer activity.

  14. Detecting adenosine triphosphate in the pericellular space.

    PubMed

    Falzoni, Simonetta; Donvito, Giovanna; Di Virgilio, Francesco

    2013-06-06

    Release of adenosine triphosphate (ATP) into the extracellular space occurs in response to a multiplicity of physiological and pathological stimuli in virtually all cells and tissues. A role for extracellular ATP has been identified in processes as different as neurotransmission, endocrine and exocrine secretion, smooth muscle contraction, bone metabolism, cell proliferation, immunity and inflammation. However, ATP measurement in the extracellular space has proved a daunting task until recently. To tackle this challenge, some years ago, we designed and engineered a novel luciferase probe targeted to and expressed on the outer aspect of the plasma membrane. This novel probe was constructed by appending to firefly luciferase the N-terminal leader sequence and the C-terminal glycophosphatidylinositol anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase, is targeted and localized to the outer side of the plasma membrane. With this probe, we have generated stably transfected HEK293 cell clones that act as an in vitro and in vivo sensor of the extracellular ATP concentration in several disease conditions, such as experimentally induced tumours and inflammation.

  15. Computer-assisted analysis of adenosine triphosphate data.

    PubMed

    Erkenbrecher, C W; Crabtree, S J; Stevenson, L H

    1976-09-01

    A computer program has been written to assist in the analysis of adenosine 5'-triphosphate data. The program is designed to calculate a dilution curve and to correct sample and adenosine 5'-triphosphate standard data for background and dilution effects. In addition, basic statistical parameters and estimates of biomass carbon are also calculated for each group of samples and printed in a convenient format. The versatility of the program to analyze data from both qauatic and terrestrial samples is noted as well as its potential use with various types of instrumentation and extraction techniques.

  16. 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

  17. Influence of 4-pyridone-3-carboxamide-1Β-D-ribonucleoside (4PYR) on activities of extracellular enzymes in endothelial human cells.

    PubMed

    Pelikant-Małecka, I; Sielicka, A; Kaniewska, E; Smoleński, R T; Słomińska, E M

    2016-12-01

    Previous studies demonstrated that human endothelial cells were capable to phosphorylate 4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) to monophosphate (4PYMP) and formed another metabolite-an analog of NAD (4PYRAD). Elevated levels of 4PYMP and 4PYRAD had an adverse effect on energy balance-depressed adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) concentration in human endothelial cells. Ecto-enzymes such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD); ecto-5'-nucleotidase (e5'NT); and ecto-adenosine deaminase (eADA) are involved in controlling of inflammation and platelet aggregation. This study aimed to evaluate influence of 4PYR and its metabolites on activities of extracellular enzymes in human endothelial cells. Endothelial cells (endothelial cell line HMEC-1) were treated with 100 uM 4PYR for 0, 24, 48, or 72 hours. After incubation, intact HMEC-1 cells were incubated with suitable substrate. Simultaneously, in another path of experiments intracellular concentration of 4PYMP and 4PYRAD had been analyzed. Conversion of extracellular nucleotides into their products and intracellular concentration of 4PYMP and 4PYRAD were measured by high performance liquid chromatography (HPLC). We demonstrated that eNTPD and e5'NT activities increase after 72 hours of cell treatment with 4PYR as compared to control (0.40 ± 0.02 versus 0.29 ± 0.02 nmol/min/mg protein; 13.3 ± 0.6 versus 8.30 ± 0.34 nmol/min/mg protein, respectively, mean ± SEM). eADA activity decreases after 24 hours of cells treatment with 4PYR as compared to control (1.55 ± 0.06 versus 1.92 ± 0.13 nmol/min/mg protein, respectively, mean ± SEM). 4PYR and its derivatives have positive effect on ecto-enzymes related with ATP degradation pathway. We conclude that these increases in extracellular enzyme activities are an adaptive response to decreased intracellular ATP and NAD arising from 4PYR uptake. These changes may protect the cells from the inflammatory

  18. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  19. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... device that measures the release of adenosine triphosphate (ATP) from platelets following aggregation. This measurement is made on platelet-rich plasma using a photometer and a luminescent firefly extract. Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  20. Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

    PubMed

    Shah, Mrugeshkumar K; Kadowitz, Philip J

    2002-01-01

    The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.

  1. 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.

  2. 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

  3. 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.

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

    PubMed

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

    2016-09-01

    Although some findings have reported the medicinal properties of Jimson weed (Datura stramonium L.), there exist some serious neurological effects such as hallucination, loss of memory and anxiety, which has been reported in folklore. Consequently, the modulatory effect of alkaloid extracts from leaf and fruit of Jimson weed on critical enzymes of the purinergic [ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5'-nucleotidase (E-NTDase), alkaline phosphatase (ALP) and Na(+)/K(+) ATPase] system of neurotransmission was the focus of this study. Alkaloid extracts were prepared by solvent extraction method and their interaction with the activities of these enzymes were assessed (in vitro) in rat brain tissue homogenate and in vivo in rats administered 100 and 200mg/kg body weight (p.o) of the extracts for thirty days, while administration of single dose (1mg/kg body weight; i.p.) of scopolamine served as the positive control. The extracts were also investigated for their Fe(2+) and Cu(2+) chelating abilities and GC-MS characterization of the extracts was also carried out. The results revealed that the extracts inhibited activates of E-NTPDase, E-NTDase and ALP in a concentration dependent manner, while stimulating the activity of Na(+)/K(+) ATPase (in vitro). Both extracts also exhibited Fe(2+) and Cu(2+) chelating abilities. Considering the EC50 values, the fruit extract had significantly higher (P<0.05) modulatory effect on the enzymes' activity as well as metal chelating abilities, compared to the leaf extract; however, there was no significant difference (P>0.05) in both extracts' inhibitory effects on E-NTDase. The in vivo study revealed reduction in the activities of ENTPDase, E-NTDase, and Na(+)/K(+) ATPase in the extract-administered rat groups compared to the control group, while an elevation in ALP activity was observed in the extract-administered rat groups compared to the control group. GC-MS characterization revealed the presence of

  5. 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

  6. 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.

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

    PubMed

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

    2012-11-21

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

  8. Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage: the neuroprotective effects of adenosine triphosphate against apoptosis.

    PubMed

    Lu, Na; Wang, Baoying; Deng, Xiaohui; Zhao, Honggang; Wang, Yong; Li, Dongliang

    2014-09-01

    After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

  9. Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage: the neuroprotective effects of adenosine triphosphate against apoptosis

    PubMed Central

    Lu, Na; Wang, Baoying; Deng, Xiaohui; Zhao, Honggang; Wang, Yong; Li, Dongliang

    2014-01-01

    After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations (2, 4, 6, 8, 10 mmol/L) over time (1, 2, 3, and 6 hours) on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SY5Y cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury. PMID:25368646

  10. 2-Selenouridine triphosphate synthesis and Se-RNA transcription.

    PubMed

    Sun, Huiyan; Jiang, Sibo; Caton-Williams, Julianne; Liu, Hehua; Huang, Zhen

    2013-09-01

    2-Selenouridine ((Se)U) is one of the naturally occurring modifications of Se-tRNAs ((Se)U-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the (Se)U-RNA via 2-selenouridine triphosphate ((Se)UTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized (Se)UTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of (Se)U-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed (Se)U-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of (Se)U-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.

  11. 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)

  12. Ubiquitination and filamentous structure of cytidine triphosphate synthase

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  13. 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. PMID:27468425

  14. Magnetite nanoparticle-induced fluorescence quenching of adenosine triphosphate-BODIPY Conjugates: application to adenosine triphosphate and pyrophosphate sensing.

    PubMed

    Yu, Cheng-Ju; Wu, Su-Mei; Tseng, Wei-Lung

    2013-09-17

    We report that magnetite nanoparticles (Fe3O4 NPs) act as an efficient quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) that is highly fluorescent in bulk solution. BODIPY-ATP molecules attached to the surface of Fe3O4 NPs through the coordination between the triphosphate group of BODIPY-ATP and Fe(3+)/Fe(2+) on the NP surface. The formed complexes induced an apparent reduction in the BODIPY-ATP fluorescence resulting from an oxidative-photoinduced electron transfer (PET) from the BODIPY-ATP excited state to an unfilled d shell of Fe(3+)/Fe(2+) on the NP surface. A comparison of the Stern-Volmer quenching constant between Fe(3+) and Fe(2+) suggests that Fe(3+) on the NP surface dominantly controls this quenching process. The efficiency for Fe3O4 NP-induced fluorescence quenching of the BODIPY-ATP was enhanced by increasing the concentration of Fe3O4 NPs and lowering the pH of the solution to below 6.0. We found that pyrophosphate and ATP compete with BODIPY-ATP for binding to Fe3O4 NPs. Thus, we amplified BODIPY-ATP fluorescence in the presence of increasing the pyrophosphate and ATP concentration; the detection limits at a signal-to-noise ratio of 3 for pyrophosphate and ATP were determined to be 7 and 30 nM, respectively. The Fe3O4 NP-based competitive binding assay detected ATP and pyrophosphate in only 5 min. The selectivity of this assay for ATP over metal ions, amino acids, and adenosine analogues is particularly high. The practicality of using the developed method to determine ATP in a single drop of blood is also validated.

  15. Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate).

    PubMed

    Hori, Mika; Suzuki, Tetsuya; Minakawa, Noriaki; Matsuda, Akira; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2011-09-01

    8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.

  16. Sustained release carrier for adenosine triphosphate as signaling molecule.

    PubMed

    Wischke, Christian; Weigel, Judith; Bulavina, Larisa; Lendlein, Andreas

    2014-12-10

    Adenosine triphosphate (ATP) is a molecule with a fascinating variety of intracellular and extracellular biological functions that go far beyond energy metabolism. Due to its limited passive diffusion through biological membranes, controlled release systems may allow to interact with ATP-mediated extracellular processes. In this study, two release systems were explored to evaluate the capacity for either long-term or short-term release: (i) Poly[(rac-lactide)-co-glycolide] (PLGA) implant rods were capable of ATP release over days to weeks, depending on the PLGA molecular weight and end-group capping, but were also associated with partial hydrolytic degradation of ATP to ADP and AMP, but not adenosine. (ii) Thermosensitive methylcellulose hydrogels with a gelation occurring at body temperature allowed combining adjustable loading levels and the capacity for injection, with injection forces less than 50N even for small 27G needles. Finally, a first in vitro study illustrated purinergic-triggered response of primary murine microglia to ATP released from hydrogels, demonstrating the potential relevance for biomedical applications.

  17. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    PubMed

    Makarchikov, Alexander F; Wins, Pierre; Janssen, Edwin; Wieringa, Bé; Grisar, Thierry; Bettendorff, Lucien

    2002-10-21

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

  18. Intracellular Adenosine Triphosphate Deprivation through Lanthanide-Doped Nanoparticles.

    PubMed

    Tian, Jing; Zeng, Xiao; Xie, Xiaoji; Han, Sanyang; Liew, Oi-Wah; Chen, Yei-Tsung; Wang, Lianhui; Liu, Xiaogang

    2015-05-27

    Growing interest in lanthanide-doped nanoparticles for biological and medical uses has brought particular attention to their safety concerns. However, the intrinsic toxicity of this new class of optical nanomaterials in biological systems has not been fully evaluated. In this work, we systematically evaluate the long-term cytotoxicity of lanthanide-doped nanoparticles (NaGdF4 and NaYF4) to HeLa cells by monitoring cell viability (mitochondrial activity), adenosine triphosphate (ATP) level, and cell membrane integrity (lactate dehydrogenase release), respectively. Importantly, we find that ligand-free lanthanide-doped nanoparticles induce intracellular ATP deprivation of HeLa cells, resulting in a significant decrease in cell viability after exposure for 7 days. We attribute the particle-induced cell death to two distinct cell death pathways, autophagy and apoptosis, which are primarily mediated via the interaction between the nanoparticle and the phosphate group of cellular ATP. The understanding gained from the investigation of cytotoxicity associated with lanthanide-doped nanoparticles provides keen insights into the safe use of these nanoparticles in biological systems.

  19. Footprint traversal by adenosine-triphosphate-dependent chromatin remodeler motor

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

    Adenosine-triphosphate (ATP)-dependent chromatin remodeling enzymes (CREs) are biomolecular motors in eukaryotic cells. These are driven by a chemical fuel, namely, ATP. CREs actively participate in many cellular processes that require accessibility of specific segments of DNA which are packaged as chromatin. The basic unit of chromatin is a nucleosome where 146 bp ˜ 50 nm of a double-stranded DNA (dsDNA) is wrapped around a spool formed by histone proteins. The helical path of histone-DNA contact on a nucleosome is also called “footprint.” We investigate the mechanism of footprint traversal by a CRE that translocates along the dsDNA. Our two-state model of a CRE captures effectively two distinct chemical (or conformational) states in the mechanochemical cycle of each ATP-dependent CRE. We calculate the mean time of traversal. Our predictions on the ATP dependence of the mean traversal time can be tested by carrying out in vitro experiments on mononucleosomes.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Fluorescence detection of adenosine triphosphate through an aptamer-molecular beacon multiple probe.

    PubMed

    Zeng, Xiaodan; Zhang, Xiaoling; Yang, Wen; Jia, Hongying; Li, Yamin

    2012-05-01

    An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

  5. Clickable 5'-γ-ferrocenyl adenosine triphosphate bioconjugates in kinase-catalyzed phosphorylations.

    PubMed

    Wang, Nan; She, Zhe; Lin, Yen-Chun; Martić, Sanela; Mann, David J; Kraatz, Heinz-Bernhard

    2015-03-23

    Clickable co-substrate: A tri-functional 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative containing a clickable site was synthesized. This compound is an effective co-substrate in kinase-catalyzed phosphorylation reactions, which can be detected by both electrochemical and immunoassay detection methods. The clickable reaction site makes direct modification possible, which greatly expands its application.

  6. 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)

  7. Determination of adenosine triphosphate on marine particulates: synthesis of methods for use on OTEC samples

    SciTech Connect

    Jones, A.T.; Hartwig, E.O.

    1982-08-01

    Adenosine triphosphate (ATP) is an indicator of living biomass in marine particulates. This report details the method used by Lawrence Berkeley Laboratory to analyze particulate ATP in samples taken from oligotrophic, tropical ocean waters. It represents a synthesis of previously published methods.

  8. Determination of Adenosine Triphosphate on Marine Particulates:Synthesis of Methods for Use on OTEC Samples

    SciTech Connect

    Jones, Anthony T.; Hartwig, Eric O.

    1982-08-01

    Adenosine triphosphate (ATP) is an indicator of living biomass in marine particulates. This report details the method used by Lawrence Berkeley Laboratory to analyze particulate ATP in samples taken from oligotrophic, tropical ocean waters. It represents a synthesis of previously published methods.

  9. Distribution of 5'-triphosphate termini on the mRNA of Escherichia coli.

    PubMed Central

    Bieger, C D; Nierlich, D P

    1989-01-01

    We have determined the distribution of 5'-nucleoside triphosphates on the RNA in Escherichia coli. These groups represent the initial nucleoside triphosphate incorporated when RNA polymerase initiates transcription. It was estimated that at least 15% of polysome-associated messengers had triphosphates. This was interpreted to mean that removal of the triphosphate or messenger leader is not necessary for the functioning of most mRNAs but that a substantial amount of messenger processing occurs in the polysome pool. We found that the ratio of GTP- to ATP-initiated messengers was about 2 to 1. Since prior work has indicated that G- and A-initiated RNAs decay at the same rate and since a compilation of messenger start sites shows an A preference, this value implies that there is a significant physiological selection of G-initiated transcripts. We also characterized the 5'-terminal groups on RNAs in other fractions. A small amount was found associated with 30S ribosomes, presumably in initiation complexes; such complexes have not previously been detected in situ. In addition, it was concluded that the 5' terminus of rRNA precursors is processed more rapidly than is implied by the current literature. PMID:2464575

  10. Hydrolysis of triphosphate from detergents in a rural waste water system.

    PubMed

    Halliwell, D J; McKelvie, I D; Hart, B T; Dunhill, R H

    2001-02-01

    The concentrations of detergent phosphates in raw sewage entering a small, predominantly domestic waste water treatment facility were determined using an ion chromatographic-flow injection analysis technique. Hourly loads of detergent phosphates were measured between 0600 and 2300 hrs (the major flow period in the plant) on days of both low and high phosphorus loads. The calculated loads of detergent phosphorus entering the plant on low and high load days were 260 g P/day and 350 g P/day, respectively. The half-life of detergent phosphates (triphosphate) in waste waters was measured to be 7.3 hours at 15 degrees C and 3.0 h at 20 degrees C. The major factor contributing to triphosphate degradation in waste water was shown to be biological in nature, with the most likely mechanism being enzymatic hydrolysis.

  11. Metabolic Cooperative Control of Electrolyte Levels by Adenosine Triphosphate in the Frog Muscle

    PubMed Central

    Gulati, J.; Ochsenfeld, M. M.; Ling, G. N.

    1971-01-01

    This study examines the effects of metabolic inhibitors on the content of cellular K, Na, and adenosine triphosphate (ATP). ATP and K are seen to fall in the inhibited tissues. The ATP content is correlated with the K content. The role of ATP is examined according to a recent biophysical approach. It is suggested that ATP may control the electrolyte levels by inducing conformational changes in the cytoplasmic proteins. PMID:5316285

  12. Microcontroller-assisted compensation of adenosine triphosphate levels: instrument and method development.

    PubMed

    Hu, Jie-Bi; Chen, Ting-Ru; Chen, Yu-Chie; Urban, Pawel L

    2015-01-30

    In order to ascertain optimum conditions for biocatalytic processes carried out in vitro, we have designed a bio-opto-electronic system which ensures real-time compensation for depletion of adenosine triphosphate (ATP) in reactions involving transfer of phosphate groups. The system covers ATP concentration range of 2-48 μM. The report demonstrates feasibility of the device operation using apyrase as the ATP-depleting enzyme.

  13. A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme.

    PubMed

    Anai, M; Mihara, T; Yamanaka, M; Shibata, T; Takagi, Y

    1975-07-01

    A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.

  14. 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

  15. Extraction and quantification of adenosine triphosphate in mammalian tissues and cells.

    PubMed

    Chida, Junji; Kido, Hiroshi

    2014-01-01

    Adenosine 5'-triphosphate (ATP) is the "energy currency" of organisms and plays central roles in bioenergetics, whereby its level is used to evaluate cell viability, proliferation, death, and energy transmission. In this chapter, we describe an improved and efficient method for extraction of ATP from tissues and cells using phenol-based reagents. The chaotropic extraction reagents reported so far co-precipitate ATP with insoluble proteins during extraction and with salts during neutralization. In comparison, the phenol-based reagents extract ATP well without the risks of co-precipitation. The extracted ATP can be quantified by the luciferase assay or high-performance liquid chromatography.

  16. 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.

  17. [An adenosine triphosphate bioluminescence assay for detecting the number of living cells].

    PubMed

    Liu, S; Peng, Z; Wang, H; Lou, J; He, B; Tang, Q; Qiu, D

    2000-06-01

    The method for detecting the number of living cells was studied. Using an adenosine triphosphate (ATP) bioluminescence assay, the present authors reported a perfect linear relationship between lg ATP concentrations and lg luminescence counts (r = 0.9963) as well as a relationship between lg number of cells and lg ATP luminescence counts (r = 0.9922). The detectable cells ranged from 10(2) to 10(6) cells/ml, the coefficients of variation 1-3%. This method is simple, accurate and sensitive and has a high reproducibility.

  18. Model Linking Plasma and Intracellular Tenofovir/Emtricitabine with Deoxynucleoside Triphosphates

    PubMed Central

    Castillo-Mancilla, Jose R.; Bushman, Lane R.; Zheng, Jia-Hua; Kiser, Jennifer J.; MaWhinney, Samantha; Anderson, Peter L.

    2016-01-01

    The coformulation of the nucleos(t)ide analogs (NA) tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC) is approved for HIV-infection treatment and prevention. Plasma TFV and FTC undergo complicated hybrid processes to form, accumulate, and retain as their active intracellular anabolites: TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP). Such complexities manifest in nonlinear intracellular pharmacokinetics (PK). In target cells, TFV-DP/FTC-TP compete with endogenous deoxynucleoside triphosphates (dNTP) at the active site of HIV reverse transcriptase, underscoring the importance of analog:dNTP ratios for antiviral efficacy. However, NA such as TFV and FTC have the potential to disturb the dNTP pool, which could augment or reduce their efficacies. We conducted a pharmacokinetics-pharmacodynamics (PKPD) study among forty subjects receiving daily TDF/FTC (300 mg/200 mg) from the first-dose to pharmacological intracellular steady-state (30 days). TFV/FTC in plasma, TFV-DP/FTC-TP and dNTPs in peripheral blood mononuclear cells (PBMC) were quantified using validated LC/MS/MS methodologies. Concentration-time data were analyzed using nonlinear mixed effects modeling (NONMEM). Formations and the accumulation of intracellular TFV-DP/FTC-TP was driven by plasma TFV/FTC, which was described by a hybrid of first-order formation and saturation. An indirect response link model described the interplay between TFV-DP/FTC-TP and the dNTP pool change. The EC50 (interindividual variability, (%CV)) of TFV-DP and FTC-TP on the inhibition of deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP) production were 1020 fmol/106 cells (130%) and 44.4 pmol/106 cells (82.5%), resulting in (90% prediction interval) 11% (0.45%, 53%) and 14% (2.6%, 35%) reductions. Model simulations of analog:dNTP molar ratios using IPERGAY dosing suggested that FTC significantly contributes to the protective effect of preexposure prophylaxis (PrEP). Simulation

  19. A new deletion in autosomal dominant guanosine triphosphate cyclohydrolase I deficiency gene--Segawa disease.

    PubMed

    Bianca, S; Bianca, M

    2006-02-01

    Hereditary Progressive Dystonia with marked diurnal fluctuation (HPD) is an autosomally dominantly inherited dystonia which is characterized by marked diurnal fluctuation of symptoms and by marked and sustained response to levodopa associated with mutations in guanosine triphosphate cyclohydrolase (GCH-1) deficiency gene. We report an italian patient with a new 18 bp deletion at 267 in exon 1 in the GCH-1 gene. The peculiarity of our patient is the new mutations never reported and mnemonic disturbances that are also not reported in the classical HPD.A genotype-phenotype relationship may be suggested between different gene mutations and non classical clinical manifestations.

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

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  1. Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases.

    PubMed

    Yang, Xiaorong; Liu, Xinran; Musser, Derek M; Moustafa, Ibrahim M; Arnold, Jamie J; Cameron, Craig E; Boehr, David D

    2017-03-03

    The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used (31)P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correct versus incorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analyses of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motifs B and D, and the nucleoside triphosphate reorientation represent separable, "tunable" fidelity checkpoints.

  2. Role of inosine triphosphate pyrophosphatase gene variant on fever incidence during zidovudine antiretroviral therapy.

    PubMed

    Coelho, A V C; Silva, S P S; Zandonà, L; Stocco, G; Decorti, G; Crovella, S

    2017-01-23

    Zidovudine, the antiretroviral drug used to treat HIV infection, commonly causes adverse effects, such as systemic fever and gastrointestinal alterations. In the present study, the potential role of inosine triphosphate pyrophosphatase (ITPA) gene variant on the incidence of adverse events during antiretroviral therapy (ART) of HIV with zidovudine was discussed. Individuals from Northeastern Brazil (N = 204) receiving treatment for HIV-1 infection were recruited. Zidovudine-related adverse effects developed during the treatment were registered. The rs1127354 polymorphism in the ITPA gene was genotyped using real-time PCR to assess whether this single nucleotide polymorphism was associated with the occurrence of zidovudine-related adverse effects. We observed a significant association between the ITPA variant genotype and the reported systemic fever (odds ratio = 7.17, 95% confidence interval = 1.19-43.15; P = 0.032). Zidovudine use could indirectly lead to an increase in the levels of inosine monophosphate in an antimetabolite-like manner, which is converted to inosine triphosphate (ITP). The rs1127354 variant caused a decrease in ITPA activity, thereby leading to ITP accumulation. This in turn resulted in cytotoxicity, which was manifested by neutropenia and fever. Therefore, we hypothesized a pharmacogenetic model involving the ITPA variant genotype in multifactorial components that act together to determine the onset of zidovudine-related adverse effects.

  3. Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis*♦

    PubMed Central

    Vorontsov, Ivan I.; Wu, Ying; DeLucia, Maria; Minasov, George; Mehrens, Jennifer; Shuvalova, Ludmilla; Anderson, Wayne F.; Ahn, Jinwoo

    2014-01-01

    EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity. PMID:24338016

  4. Thermostability of mammalian brain ribosomes and the effects of nucleoside triphosphates on their heat-sensitivity.

    PubMed

    Grove, B K; Johnson, T C; Gilbert, B E

    1974-02-01

    Mammalian brain ribosomes were found to be heat-labile. On preincubation of the ribosomes at 37 degrees C, their ability to participate in polypeptide-synthesis reactions was substantially diminished. Despite the sensitivity of ribosomal protein synthesis to heat-inactivation, preincubation resulted in no significant alterations in ribosomal sedimentation profiles or changes in the integrity of the ribosomal RNA. The thermolability of brain ribosomes was shown to be associated with their inability to bind both template RNA and aminoacyl-tRNA. Similar experiments with brain ribosomal subunits demonstrated that the small (40S) subunit was more sensitive to heat-inactivation than the large (60S) subunit. The presence of ATP (1mm) protected ribosomes from thermal inactivation, although this protection was shown to be temporary. The protection appeared to be specific to nucleoside triphosphates, since GTP and UTP also stabilized ribosomes to thermal denaturation whereas nucleoside diphosphates (ADP) and nucleoside monophosphates (AMP and cyclic AMP) did not alter ribosomal sensitivity to heat. Although 1mm concentrations of nucleoside triphosphates protected ribosomes from heat-inactivation, the presence of higher concentrations resulted in complete inactivation of ribosomal activity.

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

    PubMed Central

    Desler, Claus; Frederiksen, Jane H.; Angleys, Maria; Maynard, Scott; Keijzers, Guido; Fagerlund, Birgitte; Mortensen, Erik Lykke; Osler, Merete; Lauritzen, Martin; Bohr, Vilhelm A.; Rasmussen, Lene Juel

    2016-01-01

    Mitochondrial bioenergetics, mitochondrial reactive oxygen species (ROS) and cellular levels of nucleotides have been hypothesized as early indicators of Alzheimer’s disease (AD). Utilizing relative decline of cognitive ability as a predictor of AD risk, we evaluated the correlation between change of cognitive ability and mitochondrial bioenergetics, ROS and cellular levels of deoxyribonucleotides. Change of cognitive abilities, scored at ages of approximately 20 and 57 was determined for a cohort of 1985 male participants. Mitochondrial bioenergetics, mitochondrial ROS and whole-cell levels of deoxyribonucleotide triphosphates were measured in peripheral blood mononuclear cells (PBMCs) from a total of 103 selected participants displaying the most pronounced relative cognitive decline and relative cognitive improvement. We show that relative cognitive decline is associated with higher PBMC content of deoxythymidine-triphosphate (dTTP) (20%), but not mitochondrial bioenergetics parameters measured in this study or mitochondrial ROS. Levels of dTTP in PBMCs are indicators of relative cognitive change suggesting a role of deoxyribonucleotides in the etiology of AD. PMID:26408413

  6. Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly(iso-butylcyanoacrylate) nanocapsules.

    PubMed

    Hillaireau, H; Le Doan, T; Besnard, M; Chacun, H; Janin, J; Couvreur, P

    2006-10-31

    Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly(iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.

  7. Maltose modified poly(propylene imine) dendrimers as potential carriers of nucleoside analog 5'-triphosphates.

    PubMed

    Szulc, Aleksandra; Signorelli, Marco; Schiraldi, Alberto; Appelhans, Dietmar; Voit, Brigitte; Bryszewska, Maria; Klajnert-Maculewicz, Barbara; Fessas, Dimitrios

    2015-11-30

    Poly(propylene imine) (PPI) dendrimers contained surface maltose modification are proposed as drug carriers for nucleoside analog (NA) 5'-triphosphates. The aim of this study was to investigate the interactions between PPI dendrimers of 3rd (G3) or 4th (G4) generation and cytidine-5'-triphosphate (CTP) by Isothermal Titration Calorimetry method. CTP was used as a model molecule of pyrimidine nucleoside analog-cytarabine (ara-CTP) commonly used in leukemia treatment. Complexes of PPI dendrimers with NAs may help to overcome severe limitations of NAs associated with their low solubility and stability or resistance in cancer cells. In the present work, we evaluated stoichiometry and a mechanism of forming complexes between dendrimers and the nucleotide. Moreover, we examined the efficiency of complex formation in relation to dendrimer generations, a type of dendrimer modification with maltose residues and a type of solvent. It was observed that PPI dendrimers create complexes with CTP with high efficiency that makes them promising candidates for a drug delivery system.

  8. Rapid changes in deoxynucleoside triphosphate pools in mammalian cells treated with mutagens

    SciTech Connect

    Das, S.K.; Benditt, E.P.; Loeb, L.A.

    1983-07-29

    The rapid increase in cellular deoxynucleoside triphosphate (dNTP) concentrations in Chinese Hamster cell line V79 after exposure to known mutagens is described. With this cell line an expansion of dATP and dTTP pools was detected; changes in dCTP were not large; changes in dGTP were either not significant or too low to quantitate. This situation may reflect the existence of imbalances in dNTP pools at the DNA replication fork. The expansion of dATP and dTTP pools occurred within 2 to 4 hours after exposure of cultured cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Ultraviolet light (UV), mitomycin C, and cytosine arabinoside also caused similar dNTP pool changes.

  9. Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate.

    PubMed

    Song, Weiling; Zhang, Qiao; Xie, Xuxu; Zhang, Shusheng

    2014-11-15

    In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6 × 10(-10)M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

  10. Adenosine triphosphate acts as a paracrine signaling molecule to reduce the motility of T cells.

    PubMed

    Wang, Chiuhui Mary; Ploia, Cristina; Anselmi, Fabio; Sarukhan, Adelaida; Viola, Antonella

    2014-06-17

    Organization of immune responses requires exchange of information between cells. This is achieved through either direct cell-cell contacts and establishment of temporary synapses or the release of soluble factors, such as cytokines and chemokines. Here we show a novel form of cell-to-cell communication based on adenosine triphosphate (ATP). ATP released by stimulated T cells induces P2X4/P2X7-mediated calcium waves in the neighboring lymphocytes. Our data obtained in lymph node slices suggest that, during T-cell priming, ATP acts as a paracrine messenger to reduce the motility of lymphocytes and that this may be relevant to allow optimal tissue scanning by T cells.

  11. Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Dubois-Colas, Nicolas; Petit-Jentreau, Laetitia; Barreiro, Luis B; Durand, Sylvère; Soubigou, Guillaume; Lecointe, Cécile; Klibi, Jihène; Rezaï, Keyvan; Lokiec, François; Coppée, Jean-Yves; Gicquel, Brigitte; Tailleux, Ludovic

    2014-09-01

    Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

  12. A Destabilized Case of Stable Effort Angina Pectoris Induced by Low-dose Adenosine Triphosphate.

    PubMed

    Sueta, Daisuke; Kojima, Sunao; Izumiya, Yasuhiro; Yamamuro, Megumi; Kaikita, Koichi; Hokimoto, Seiji; Ogawa, Hisao

    A 79-year-old man was diagnosed with sudden deafness. He had previously experienced a suspected episode of angina pectoris. At a local hospital, after 500 mg of hydrocortisone and 80 mg adenosine triphosphate (ATP) were administered, he became aware of chest discomfort. An electrocardiogram revealed serious ST-segment depressions. He was diagnosed with a non-ST elevated myocardial infarction (NSTEMI). Emergency coronary angiography revealed triple vessel disease, and the lesion was successfully stented. The mechanisms whereby the stable effort angina pectoris destabilized in this case were thought to include a reduction of the local blood flow because of an ATP product and probable thrombus formation in response to the administered steroids.

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

    PubMed

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

    2015-03-09

    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.

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

    PubMed

    Bogolyubov, Dmitry

    2007-01-01

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

  15. A Destabilized Case of Stable Effort Angina Pectoris Induced by Low-dose Adenosine Triphosphate

    PubMed Central

    Sueta, Daisuke; Kojima, Sunao; Izumiya, Yasuhiro; Yamamuro, Megumi; Kaikita, Koichi; Hokimoto, Seiji; Ogawa, Hisao

    2016-01-01

    A 79-year-old man was diagnosed with sudden deafness. He had previously experienced a suspected episode of angina pectoris. At a local hospital, after 500 mg of hydrocortisone and 80 mg adenosine triphosphate (ATP) were administered, he became aware of chest discomfort. An electrocardiogram revealed serious ST-segment depressions. He was diagnosed with a non-ST elevated myocardial infarction (NSTEMI). Emergency coronary angiography revealed triple vessel disease, and the lesion was successfully stented. The mechanisms whereby the stable effort angina pectoris destabilized in this case were thought to include a reduction of the local blood flow because of an ATP product and probable thrombus formation in response to the administered steroids. PMID:27853071

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

    PubMed

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

    2012-01-31

    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, (24)Mg, and (25)Mg, 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)].

  17. Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular.

    PubMed

    Bodin, P; Burnstock, G

    2001-12-01

    In response to increased shear stress, vascular endothelial cells release adenosine triphosphate (ATP) by an unknown mechanism. We have investigated this mechanism using different approaches. First, we discovered that quinacrine, used to locate intracellular stores of ATP bound to peptides, displayed a granular fluorescence, typical of vesicular storage. Second, we found that two inhibitors of vesicular transport (monensin and N-ethylmaleimide) produced a highly significant reduction in the release of ATP from vascular endothelial cells in response to increased shear stress. Preliminary experiments using inhibitors of the cystic fibrosis transmembrane regulator, the sulfonylurea receptor, and the multidrug resistance protein showed no involvement of these ATP-binding cassette transporter proteins (previously characterized in endothelial cells) in the mechanism of release of ATP. We suggest, therefore, that the release of ATP from vascular endothelial cells, like that of nerve cells, is probably by vesicular exocytosis.

  18. The breakdown of adenosine triphosphate in the contraction cycle of the frog sartorius muscle

    PubMed Central

    Mommaerts, W. F. H. M.; Wallner, A.

    1967-01-01

    1. It is confirmed that a fluorodinitrobenzene (FDNB)-treated frog sartorius muscle does not split phosphorylcreatine in the course of its contraction cycle, but does use adenosine triphosphate (ATP). 2. Good stoicheiometric relations between the diminution of ATP and the formation of adenosine diphosphate (ADP), adenosine monophosphate (AMP) and phosphate are obtained, and in a 0·2 sec tetanus at 0° C the net break-down of ATP amounts to 0·27, the total equivalent break-down to 0·34 μmoles/g. 3. There is no difference in this quantity between muscles interrupted at the height of contraction and those that have also relaxed, and, in experiments specifically designed to determine relaxation metabolism separately, no such metabolism is found. Thus, all the ATP-break-down occurs in the contraction phase. PMID:6065882

  19. 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".

  20. 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

  1. Fabrication of Adenosine Triphosphate-Molecule Recognition Chip by Means of Bioluminous Enzyme Luciferase

    NASA Astrophysics Data System (ADS)

    Tanii, Takashi; Goto, Tomomi; Iida, Tomoyuki; Koh-Masahara, Meishoku; Ohdomari, Iwao

    2001-10-01

    We have succeeded in detecting the adenosine triphosphate (ATP) concentration in a solution quantitatively using an ATP-molecule recognition chip. The ATP-molecule recognition chip is composed of a silicon photodiode on which bioluminous enzyme luciferase is immobilized. When the chip was immersed in an ATP-containing solution, the luciferase emitted light and the photoinduced current detected by the photodiode was in proportion to the ATP concentration. We found that the photoinduced current fits the Michaelis-Menten plot. These results indicate that the luciferase is successfully immobilized on the silicon chip without losing the bioluminous activity and that the proposed device enables us to detect the ATP concentration in a solution by measuring the photoinduced current.

  2. 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.

  3. Thiamine triphosphate synthesis in rat brain occurs in mitochondria and is coupled to the respiratory chain.

    PubMed

    Gangolf, Marjorie; Wins, Pierre; Thiry, Marc; El Moualij, Benaïssa; Bettendorff, Lucien

    2010-01-01

    In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P(i). This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H(+) channel of F(0)F(1)-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Deltap) is required. Collapsing Deltap after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H(+)-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P(i). However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.

  4. Sample preparation and high-performance liquid chromatographic analysis of deoxyribonucleoside triphosphates in individual rat embryos.

    PubMed

    Mole, M L; Hunter, D L; Gao, P; Lau, C

    1998-06-01

    A rapid, robust, and sensitive method has been developed to measure concentrations of deoxyribonucleoside triphosphates in individual, day 14 rat embryos by modifying and optimizing existing methods for cellular extracts. Significant changes include: (i) oxidative degradation of ribonucleoside triphosphates using methylamine at lower pH (decreased from 6.5 to 4.0) to improve poor HPLC peak shape of early eluting nucleotides; (ii) glass fiber disc solid-phase extraction of the reaction mixture, which dramatically reduces impurities that interfere with nucleotide measurement, eliminates the necessity of column regeneration, and allows mobile phase recycling; and (iii) lower ionic strength (reduced from 0.4 to 0.26 or 0.12 M ammonium phosphate) and higher pH (increased from 3.25 to 5.55 or 6.98, respectively) mobile phase, conditions which are less destructive to the column's bonded phase and silica support, thereby contributing to longer column life. Enhancements include: (i) filtration of the sample prior to HPLC injection and addition of an in-line filter, guard column, and saturating precolumn of silica in the mobile phase flow, which aids substantially in extending column life and improves chromatographic stability, and (ii) inclusion of an internal standard to correct for mechanical losses. Limits of determination at a signal to noise ratio of 6:1 range from 5.5 to 12 pmol on-column or 0.41 to 0.87 pmol/mg of embryonic tissue depending on the specific nucleotide. Recoveries are quantitative for all nucleotides, and interassay variabilities are between 5 and 7% when quantified by peak height. The method has also been applied successfully to analysis of murine erythroleukemic cell cultures and this, when coupled with the embryo results, suggests its general utility.

  5. Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity.

    PubMed

    Knappenberger, Andrew J; Ahmad, Md Faiz; Viswanathan, Rajesh; Dealwis, Chris G; Harris, Michael E

    2016-10-18

    Class I ribonucleotide reductase (RR) maintains balanced pools of deoxyribonucleotide substrates for DNA replication by converting ribonucleoside diphosphates (NDPs) to 2'-deoxyribonucleoside diphosphates (dNDPs). Binding of deoxynucleoside triphosphate (dNTP) effectors (ATP/dATP, dGTP, and dTTP) modulates the specificity of class I RR for CDP, UDP, ADP, and GDP substrates. Crystal structures of bacterial and eukaryotic RRs show that dNTP effectors and NDP substrates bind on either side of a flexible nine-amino acid loop (loop 2). Interactions with the effector nucleobase alter loop 2 geometry, resulting in changes in specificity among the four NDP substrates of RR. However, the functional groups proposed to drive specificity remain untested. Here, we use deoxynucleoside analogue triphosphates to determine the nucleobase functional groups that drive human RR (hRR) specificity. The results demonstrate that the 5-methyl, O4, and N3 groups of dTTP contribute to specificity for GDP. The O6 and protonated N1 of dGTP direct specificity for ADP. In contrast, the unprotonated N1 of adenosine is the primary determinant of ATP/dATP-directed specificity for CDP. Structural models from X-ray crystallography of eukaryotic RR suggest that the side chain of D287 in loop 2 is involved in binding of dGTP and dTTP, but not dATP/ATP. This feature is consistent with experimental results showing that a D287A mutant of hRR is deficient in allosteric regulation by dGTP and dTTP, but not ATP/dATP. Together, these data define the effector functional groups that are the drivers of human RR specificity and provide constraints for evaluating models of allosteric regulation.

  6. 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.

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

    PubMed

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

    2013-11-08

    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.

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

    PubMed Central

    Rocha, Jeová Nina

    2016-01-01

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

  9. 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

  10. 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

  11. 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.

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

    PubMed

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

    2015-04-07

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

  14. 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

  15. Galactosylated chitosan oligosaccharide nanoparticles for hepatocellular carcinoma cell-targeted delivery of adenosine triphosphate.

    PubMed

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

    2013-07-29

    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.

  16. Homogeneously ultrasensitive electrochemical detection of adenosine triphosphate based on multiple signal amplification strategy.

    PubMed

    Chen, Xiaojun; Ge, Lingna; Guo, Buhua; Yan, Ming; Hao, Ning; Xu, Lin

    2014-08-15

    An ultrasensitive electrochemical aptasensor was successfully fabricated for the detection of adenosine triphosphate (ATP). For the first time, one detection system combined several elements: magnetic aptamer sequences for target recognition and separation, a DNAzyme assisted cyclic signal amplification strategy, layer-by-layer (LBL) quantum dots (QDs) composites for promoting square wave anodic stripping voltammetric (SWASV) analysis and Bi, Nafion (Nf) and three-dimensional ordered macroporous polyaniline-ionic liquid (Bi/Nf/3DOM PANI-IL) film modified glassy carbon electrode (GCE) for monitoring enhanced SWASV signal. The modification of Nf/3DOM PANI-IL on GCE showed that the preconcentration efficiency was improved by the electrostatic absorption of Cd(2+) with negative Nf layer with the enhanced analytical sensitivity due to a large active surface area of 3DOM structure. The increased SWASV peak current values of the label (CdS)4@SiO2 composites were found to be proportional to the logarithmic value of ATP concentrations in the range of 1pM-10nM and 10nM-1µM, with the detection limit as low as 0.5pM. The proposed aptasensor has shown an excellent performance such as high sensitivity, good selectivity and analytical application in real samples. The results demonstrated that the multiple signal amplified strategy we developed was feasible for clinical ATP assay and would provide a promising model for the detection of other small molecules.

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

    PubMed

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

    2014-03-01

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

  18. A target-triggered strand displacement reaction cycle: the design and application in adenosine triphosphate sensing.

    PubMed

    Cheng, Sheng; Zheng, Bin; Wang, Mozhen; Lam, Michael Hon-Wah; Ge, Xuewu

    2014-02-01

    A strand displacement reaction (SDR) system that runs solely on oligonucleotides has been developed for the amplification detection of adenosine triphosphate (ATP). It involves a target-induced SDR and an entropy-driven catalytic cycle of two SDRs with five oligonucleotides, denoted as substrate, fuel, catalyst, C-1, and C-2. Catalyst, released from the ATP aptamer-catalyst duplex by ATP molecule, catalyzes the SDRs to finally form the substrate-fuel duplex. All of the intermediates in the catalytic SDR processes have been identified by polyacrylamide gel electrophoresis (PAGE) analysis. The introduction of ATP into the SDR system will induce the ATP aptamer to form G-quadruplex conformation so as to release catalyst and trigger the SDR cycle. When the substrate and C-2 oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibited a "turn-on" response for ATP. The condition for detecting ATP, such as Mg²⁺ concentration, has been optimized to afford a detection limit of 20 nM. This work provides an enzyme-free biosensing strategy and has potential application in aptamer-based biosensing.

  19. Aptamer-based electrochemical biosensor for detection of adenosine triphosphate using a nanoporous gold platform.

    PubMed

    Kashefi-Kheyrabadi, Leila; Mehrgardi, Masoud A

    2013-12-01

    In spite of the promising applications of aptamers in the bioassays, the development of aptamer-based electrochemical biosensors with the improved limit of detection has remained a great challenge. A strategy for the amplification of signal, based on application of nanostructures as platforms for the construction of an electrochemical adenosine triphosphate (ATP) aptasensor, is introduced in the present manuscript. A sandwich assay is designed by immobilizing a fragment of aptamer on a nanoporous gold electrode (NPGE) and its association to second fragment in the presence of ATP. Consequently, 3, 4-diaminobenzoic acid (DABA), as a molecular reporter, is covalently attached to the amine-label of the second fragment, and the direct oxidation signal of DABA is followed as the analytical signal. The sensor can detect the concentrations of ATP as low as submicromolar scales. Furthermore, 3.2% decrease in signal is observed by keeping the aptasensor at 4 °C for a week in buffer solution, implying a desirable stability. Moreover, analog nucleotides, including GTP, UTP and CTP, do not show serious interferences and this sensor easily detects its target in deproteinized human blood plasma.

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

    PubMed

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

    2015-01-01

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

  1. 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.

  2. Improving environmental cleaning in clinical areas: staff education based on adenosine triphosphate readings.

    PubMed

    Villanueva, Ariadna; Guanche, Humberto

    2016-11-01

    Aim To describe the effect of education on environmental cleaning in patient care areas using adenosine triphosphate (ATP) readings. Method A quality improvement initiative was developed in a community hospital in Qatar. Over a two-month period, an infection-control practitioner monitored ATP readings in patient care areas, at any time and regardless of the time of the previous disinfection. The initiative included staff education, use of ATP readings and the drawing up of quarterly quality reports. The ATP readings were considered 'pass', meaning well cleaned, or 'fail', meaning non-cleaned, according to the following standards:>250 relative light units (RLU) in non-critical units and<200RLU for critical units. The proportion of test passes was calculated per 100 tests performed. Results A total of 1,617 tests were performed, after which 1,259 (78%) surfaces were identified as well cleaned. The lowest proportion of non-pass and higher ATP readings was observed in non-critical areas. The test points with the lowest proportion of passes were telephones (40.5%), a medication dispensing system (58.5%), an oximeter (66.7%) and callbox buttons (67.6%). A sustained increase in test passes was observed during the study period. Conclusion There was an improvement in environmental cleaning due to monitoring of ATP on surfaces and staff education.

  3. A multifunctional label-free electrochemical impedance biosensor for Hg(2+), adenosine triphosphate and thrombin.

    PubMed

    Chen, Lifen; Chen, Zhong-Ning

    2015-01-01

    A multifunctional label-free biosensor for the detection of Hg(2+), adenosine triphosphate and thrombin has been developed based on the changing of the electrochemical impedance spectroscopy (EIS) from the modified electrodes when nucleic acid subunits interacting with different targets. The modified electrode consists of three interaction sections, including DNA with T-T mismatch recognizing Hg(2+) to form T-Hg(2+)-T complex, split DNA chip against ATP, and DNA domin against thrombin to form G-quadruplex. Upon DNA interaction with thrombin or ATP, an increased charge transfer resistance (Rct) had been detected. However, a decreased Rct against Hg(2+) was obtained. The Rct difference (ΔRct) has relationship with the concentration of the different targets, Hg(2+), ATP and thrombin can be selectively detected with the detection limit of 0.03, 0.25, and 0.20 nmol L(-1), respectively. To separately detect the three analytes existing in the same sample, ATP aptamer, G-rich DNA strands and EDTA were applied to mask ATP, Hg(2+) or thrombin separately.

  4. Electrochemiluminescence aptasensor for adenosine triphosphate detection using host-guest recognition between metallocyclodextrin complex and aptamer.

    PubMed

    Chen, Hong; Chen, Qiong; Zhao, Yingying; Zhang, Fan; Yang, Fan; Tang, Jie; He, Pingang

    2014-04-01

    A sensitive and label-free electrochemiluminescence (ECL) aptasensor for the detection of adenosine triphosphate (ATP) was successfully designed using host-guest recognition between a metallocyclodextrin complex, i.e., tris(bipyridine)ruthenium(II)-β-cyclodextrin [tris(bpyRu)-β-CD], and an ATP-binding aptamer. In the protocol, the NH2-terminated aptamer was immobilized on a glassy carbon electrode (GCE) by a coupling interaction. After host-guest recognition between tris(bpyRu)-β-CD and aptamer, the tris(bpyRu)-β-CD/aptamer/GCE produced a strong ECL signal as a result of the photoactive properties of tris(bpyRu)-β-CD. However, in the presence of ATP, the ATP/aptamer complex was formed preferentially, which restricted host-guest recognition, and therefore less tris(bpyRu)-β-CD was attached to the GCE surface, resulting in an obvious decrease in the ECL intensity. Under optimal determination conditions, an excellent logarithmic linear relationship between the ECL decrease and ATP concentration was obtained in the range 10.0-0.05 nM, with a detection limit of 0.01 nM at the S/N ratio of 3. The proposed ECL-based ATP aptasensor exhibited high sensitivity and selectivity, without time-consuming signal-labeling procedures, and is considered to be a promising model for detection of aptamer-specific targets.

  5. 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.

  6. Double-functionalized gold nanoparticles with split aptamer for the detection of adenosine triphosphate.

    PubMed

    Cheng, Sheng; Zheng, Bin; Wang, Mozhen; Lam, Michael Hon-Wah; Ge, Xuewu

    2013-10-15

    A newly designed functionalization type for gold nanoparticles (AuNP) with split aptamer has been developed for the detection of adenosine triphosphate (ATP). The ATP aptamer was split into two parts with their 5' prime or 3' prime modified with thiol. Both the 5' SH and 3' SH modified strands for each split aptamer fragment were functionalized onto the same AuNP to construct double-functionalized AuNP-DNA conjugates. Thus, the split aptamer can be reassembled into intact folded structure in the presence of ATP molecule with two potential assembly types, which induces the assembly of AuNP-DNA conjugates. In this double-functionalized system, the traditional assembly type might facilitate another assembly type, which was found to give much higher LSPR change in the presence of ATP than the traditional assembly type, and improve the sensitivity for ATP detection. Time courses of the assemble processes with different assembly types, Mg(2+) concentrations, and aptamer fragments densities on AuNP were followed using the absorption ratio at 650 nm and 520 nm. ATP response with this newly designed system was investigated using absorption spectra and dynamic light scattering method.

  7. Red blood cells (RBCs), epoxyeicosatrienoic acids (EETs) and adenosine triphosphate (ATP).

    PubMed

    Jiang, Houli; Anderson, Gail D; McGiff, John C

    2010-01-01

    In addition to serving as carriers of O(2), red blood cells (RBCs) regulate vascular resistance and the distribution of microvascular perfusion by liberating adenosine triphosphate (ATP) and epoxyeicosatrienoic acids (EETs) upon exposure to a low O(2) environment. Therefore, RBCs act as sensors that respond to low pO(2) by releasing millimolar amounts of ATP, a signaling molecule, and lipid mediators (EETs). The release of EETs occurs by a mechanism that is activated by ATP stimulation of P2X(7) receptors coupled to ATP transporters, which should greatly amplify the circulatory response to ATP. RBCs are reservoirs of EETs and the primary sources of plasma EETs, which are esterified to the phospholipids of lipoproteins. Levels of free EETs in plasma are low, about 3% of circulating EETs. RBC EETs are produced by direct oxidation of arachidonic acid (AA) esterified to glycerophospholipids and the monooxygenase-like activity of hemoglobin. On release, EETs affect vascular tone, produce profibrinolysis and dampen inflammation. A soluble epoxide hydrolase (sEH) regulates the concentrations of RBC and vascular EETs by metabolizing both cis- and trans-EETs to form dihydroxyeicosatrienoic acids (DHETs). The function and pathophysiological roles of trans-EETs and erythro-DHETs has yet to be integrated into a physiological and pathophysiological context.

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

    PubMed Central

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

    2016-01-01

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

  9. 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%.

  10. Using silicon nanowire devices to detect adenosine triphosphate liberated from electrically stimulated HeLa cells.

    PubMed

    Chen, C C; Chen, Y-Z; Huang, Y-J; Sheu, J-T

    2011-01-15

    In this study, we used a biosensor chip featuring Abl tyrosine kinase-modified silicon nanowire field-effect transistors (SiNW-FETs) to detect adenosine triphosphate (ATP) liberated from HeLa cells that had been electrically stimulated. Cells that are cultured in high-ionic-strength media or buffer environments usually undermine the sensitivity and selectively of SiNW-FET-based sensors. Therefore, we first examined the performance of the biosensor chip incorporating the SiNW-FETs in both low- and high-ionic-strength buffer solutions. Next, we stimulated, using a sinusoidal wave (1.0 V, 50 Hz, 10 min), HeLa cells that had been cultured on a cell-culture chip featuring interdigitated electrodes. The extracellular ATP concentration increased by ca. 18.4-fold after electrical stimulation. Finally, we detected the presence of extracellular ATP after removing a small amount of buffer solution from the cell-cultured chip and introducing it into the biosensor chip.

  11. Biphasic Elimination of Tenofovir Diphosphate and Nonlinear Pharmacokinetics of Zidovudine Triphosphate in a Microdosing Study

    PubMed Central

    Chen, Jianmeng; Flexner, Charles; Liberman, Rosa G.; Skipper, Paul L.; Louissaint, Nicolette; Tannenbaum, Steven R.; Hendrix, Craig; Fuchs, Edward

    2012-01-01

    Objective Phase 0 studies can provide initial pharmacokinetics (PK) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of two antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. Study Design We administered a microdose (100 μg) of 14C-labeled drug (ZDV or tenofovir disoproxil fumarate (TDF)) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in PBMCs and CD4+ cells were measured by AMS. Results The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 μg to 300 mg), while the intracellular TFV-DP PK were linear over the same dose range. ZDV-TP concentrations were lower in CD4+ cells versus total peripheral blood mononuclear cells (PBMCs), while TFV-DP concentrations were not different in CD4+ cells and PBMCs. Conclusion Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. AMS shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs. PMID:23187888

  12. 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.

  13. Effect of adenosine triphosphate and some derivatives on cerebral blood flow and metabolism.

    PubMed Central

    Forrester, T; Harper, A M; MacKenzie, E T; Thomson, E M

    1979-01-01

    1. Responses of cerebral blood vessels to peri- and intravascular doses of ATP (adenosine triphosphate) and some derivatives were studied in cat and baboon. 2. Perivascular application of ATP to cat pial arterioles gave a threshold dilatory effect at a concentration of 10(-11) M. This figure is comparable to the amount of ATP calculated to be released from electrically stimulated brain slices. 3. It is concluded that adenine nucleotides have a major role to play in the local control of cerebral blood flow. 4. Intracarotid injection of ATP showed a calculated threshold effect at 4 x 10(8) M in the cat and 4 x 10(-9) M in the baboon. 5. The threshold response of the vasculature to intracarotid adenosine lay between 4 x 10(-7) M and 4 x 10(-6) M in the baboon. Little effect was produced with AMP, pyrophosphate and inorganic phosphate. 6. Intracarotid ATP increased the oxygen consumption of the baboon brain parenchyma. This effect was attributed in part to an elevation of the cellular cyclic AMP levels. 7. Osmotic disruption of the blood-brain barrier in baboon did not affect the vasodilatory or metabolic effect of intracarotid ATP. 8. It is postulated that circulating purine compounds mediate a form of metabolic communication inthe body. Also, release of purine compounds from active local nerves might influence cerebral blood flow. PMID:119042

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

    PubMed

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

    2016-09-09

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

  15. Bond cleavages of adenosine 5'-triphosphate induced by monochromatic soft X-rays

    NASA Astrophysics Data System (ADS)

    Fujii, K.; Narita, A.; Yokoya, A.

    2014-04-01

    To investigate which type of bond is likely to be cleaved by soft X-ray exposure to an adenosine 5'-triphosphate (ATP), we observed spectral changes in X-ray absorption near edge structure (XANES) around nitrogen and oxygen K-edge of an ATP film by soft X-ray irradiation. Experiments were performed at a synchrotron soft X-ray beamline at SPring-8, Japan. The XANES spectra around the nitrogen and oxygen .K-edge slightly varied by exposure to 560 eV soft X-rays. These changes are originated from the cleavage of C-N bonds between a sugar and a nucleobase site and of C-O, P-O or O-H bond of sugar and phosphate site. From the comparison between the change in XANES intensity of σ* peak at nitrogen and that at oxygen K-edges, it is inferred that the C-O, P-O or O-H bond of sugar and phosphate is much efficiently cleaved than the C-N of N-glycoside bond by the exposure of 560 eV soft X-ray to ATP film.

  16. [Nucleoside-5'-triphosphate hydrolysis in the liver and kidney of rats with chronic alloxan diabetes].

    PubMed

    Rusina, I M; Makarchikov, A F; Makar, E A; Kubyshin, V L

    2006-01-01

    Activity and some properties of a soluble enzyme hydrolyzing nucleoside-5'-triphosphates were studied in the liver and kidney of normal and diabetic rats. The enzyme activity was shown to be reduced by 34% (p < 0.01) in the liver extracts of diabetic animals, while no difference was observed in the kidney. When ITP was used as substrate, the apparent Michaelis constant of the enzyme was significantly lower in the liver of controls as compared to experimental rats (32.3 +/- 1.3 microM and 54.3 +/- 1.0 microM, respectively, p < 0.01). The KM values of the enzyme in the kidney were not distinguishable in both groups. NTPase exhibits maximal activity at pH 7.0 and has a broad substrate specificity with respect to different nucleoside-5'-tri- and diphosphates. Molecular mass of the enzyme was estimated by gel filtration to be 63.7 +/- 0.9 kD.

  17. 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

  18. Colorimetric sensor for triphosphates and their application as a viable staining agent for prokaryotes and eukaryotes.

    PubMed

    Ghosh, Amrita; Shrivastav, Anupama; Jose, D Amilan; Mishra, Sanjiv K; Chandrakanth, C K; Mishra, Sandhya; Das, Amitava

    2008-07-15

    The chromogenic complex 1 x Zn (where 1 is (E)-4-(4-dimethylamino-phenylazo)-N,N-bispyridin-2-ylmethyl-benzenesulfonamide) showed high affinity toward the phosphate ion in tetrabutylammonium phosphate in acetonitrile solution and could preferentially bind to adenosine triphosphate (ATP) in aqueous solution at physiological pH. This binding caused a visual change in color, whereas no such change was noticed with other related anions (adenosine monophosphate, adenosine diphosphate, pyrophosphate, and phosphate) of biological significance. Thus, 1 x Zn could be used as a staining agent for different biological cells through binding to the ATP, generated in situ by the mitochondria (in eukaryotes). For prokaryotes (bacteria) the cell membrane takes care of the cells' energy conversion, since they lack mitochondria. ATP is produced in their unique cell structure on the cell membrane, which is not found in any eukaryotes. These stained cells could be viewed with normal light microscopy. This reagent could even be used for distinguishing the gram-positive and the gram-negative bacteria (prokaryotes). This dye was found to be nonlipophilic in nature and nontoxic to living microbes (eukaryotes and prokaryotes). Further, stained cells were found to grow in their respective media, and this confirmed the maintenance of viability of the microbes even after staining, unlike with many other dyes available commercially.

  19. Vascular CD39/ENTPD1 Directly Promotes Tumor Cell Growth by Scavenging Extracellular Adenosine Triphosphate12

    PubMed Central

    Feng, Lili; Sun, Xiaofeng; Csizmadia, Eva; Han, Lihui; Bian, Shu; Murakami, Takashi; Wang, Xin; Robson, Simon C; Wu, Yan

    2011-01-01

    Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5′-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X7 receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X7 expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management. PMID:21390184

  20. Digoxin and Adenosine Triphosphate Enhance the Functional Properties of Tissue-Engineered Cartilage

    PubMed Central

    Makris, Eleftherios A.; Huang, Brian J.; Hu, Jerry C.; Chen-Izu, Ye

    2015-01-01

    Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca2+-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca2+ modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10–14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52–110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca2+ imaging experiments revealed that both digoxin and ATP were able to increase Ca2+ oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca2+ modulators. PMID:25473799

  1. 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.

  2. Characterization of oxidized guanosine 5'-triphosphate as a viable inhibitor of soluble guanylyl cyclase.

    PubMed

    Bolin, Celeste; Cardozo-Pelaez, Fernando

    2009-03-15

    The guanine base is prone to oxidation by free radicals regardless of the cellular moiety it is bound to. However, under conditions of oxidative stress, 8-oxoguanosine triphosphate (oxo(8)GTP) formation has been shown to occur without oxidation of the guanine base in DNA. In vitro studies have suggested that oxo(8)GTP could impact G-protein signaling and RNA synthesis. Whether increased levels of oxo(8)GTP translate into cellular malfunction is unknown. Data presented herein show that oxo(8)GTP is formed in cell-free preparations as well as in PC12 cells after exposure to physiologically relevant oxidative conditions generated with 10 microM copper sulfate and 1 mM L-ascorbic acid (Cu/Asc). We also determined that oxo(8)GTP has biological activity as a potent inhibitor of nitric oxide-stimulated soluble guanylyl cyclase (sGC). The increase in oxo(8)GTP formation in purified GTP and PC12 cells exposed to Cu/Asc caused a significant reduction in the product of sGC activity, cGMP. This oxidation of GTP was attenuated by the addition of reduced glutathione under these same Cu/Asc conditions, thus preventing the decrease in sGC activity. This suggests that oxo(8)GTP is produced by free radicals in vivo and could have significant impact on cell functions regulated by sGC activity such as synaptic plasticity in the central nervous system.

  3. 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.

  4. 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.

  5. 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.

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

    DOE PAGES

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; ...

    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. Enzyme-based field-effect transistor for adenosine triphosphate (ATP) sensing.

    PubMed

    Migita, Satoshi; Ozasa, Kazunari; Tanaka, Tomoya; Haruyama, Tetsuya

    2007-01-01

    Adenosine triphosphate (ATP) not only functions as an energy-carrier substance and an informative molecule, but also acts as a marker substance in studies of both bio-traces and cellular/tissular viability. Due to the importance of the ATP function for living organisms, in situ assays of ATP are in demand in various fields, e.g., hygiene. In the present study, we developed an ATP sensor that combines the selective catalytic activity of enzyme and the properties of an ion selective field effect transistor (ISFET). In this system, the ATP hydrolyrase, "apyrase (EC 3.6.1.5.)" is encased in a gel and mounted on a Ta(2)O(5) ISFET gate surface. When the enzyme layer selectively catalyzes the dephosphorylation of ATP, protons are accumulated at the gate because the enzymatic reaction produces H(+) as a byproduct. Based on the interfacial enzymatic reaction, the response from the ISFET is completely dependent upon the ATP concentration in the bulk solution. This device is readily applicable to practical in situ ATP measurement, e.g. hygienic usage.

  8. Light scattering change precedes loss of cerebral adenosine triphosphate in a rat global ischemic brain model.

    PubMed

    Kawauchi, Satoko; Sato, Shunichi; Ooigawa, Hidetoshi; Nawashiro, Hiroshi; Ishihara, Miya; Kikuchi, Makoto

    2009-08-14

    Measurement of intrinsic optical signals (IOSs) is an attractive technique for monitoring tissue viability in brains since it enables noninvasive, real-time monitoring of morphological characteristics as well as physiological and biochemical characteristics of tissue. We previously showed that light scattering signals reflecting cellular morphological characteristics were closely related to the IOSs associated with the redox states of cytochrome c oxidase in the mitochondrial respiratory chain. In the present study, we examined the relationship between light scattering and energy metabolism. Light scattering signals were transcranially measured in rat brains after oxygen and glucose deprivation, and the results were compared with concentrations of cerebral adenosine triphosphate (ATP) measured by luciferin-luciferase bioluminescence assay. Electrophysiological signal was also recorded simultaneously. After starting saline infusion, EEG activity ceased at 108+/-17s, even after which both the light scattering signal and ATP concentration remained at initial levels. However, light scattering started to change in three phases at 236+/-15s and then cerebral ATP concentration started to decrease at about 260s. ATP concentration significantly decreased during the triphasic scattering change, indicating that the start of scattering change preceded the loss of cerebral ATP. The mean time difference between the start of triphasic scattering change and the onset of ATP loss was about 24s in the present model. DC potential measurement showed that the triphasic scattering change was associated with anoxic depolarization. These findings suggest that light scattering signal can be used as an indicator of loss of tissue viability in brains.

  9. Adenosine triphosphate bioluminescence analysis for rapid screening of microbial contamination in non-sterile pharmaceutical samples.

    PubMed

    Jimenez, Luis

    2004-01-01

    An Adenosine Triphosphate (ATP) bioluminescence system was compared and validated against standard methods for rapid microbiological monitoring of several non-sterile pharmaceutical formulations such as creams, tablets, and capsules. Results obtained using 1%, 2.5%, and 10% of product suspensions indicated that most samples that did not contain non-microbial ATP neither inhibited the bioluminescence reaction nor did something else. Ten percent product suspensions were inoculated with different concentrations of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Candida albicans, and Aspergillus niger. Samples were incubated for 24-120 h at 35 degrees C with shaking. Results indicated a strong inhibitory effect of microbial growth, as no microorganisms were detected by using the ATP bioluminescence assay. However, when 1% and 2.5% product suspensions were spiked with the same microorganisms, positive detection was confirmed. After incubation, all microorganisms were detected by the bioluminescence system within 24-72 h. All positive samples were confirmed by using standard plating media. However, to optimize detection of all microorganisms, different enrichment media were developed.

  10. An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells.

    PubMed

    Chida, Junji; Yamane, Kazuhiko; Takei, Tunetomo; Kido, Hiroshi

    2012-05-21

    Firefly bioluminescence is widely used in the measurement of adenosine 5'-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin-luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris-EDTA-saturated phenol (phenol-TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol-TE extraction procedure for ATP measurement in tissues and cells by luciferase assay.

  11. 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

  12. Digoxin and adenosine triphosphate enhance the functional properties of tissue-engineered cartilage.

    PubMed

    Makris, Eleftherios A; Huang, Brian J; Hu, Jerry C; Chen-Izu, Ye; Athanasiou, Kyriacos A

    2015-03-01

    Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca(2+)-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca(2+) modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10-14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52-110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca(2+) imaging experiments revealed that both digoxin and ATP were able to increase Ca(2+) oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca(2+) modulators.

  13. 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

  14. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    PubMed Central

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (–PO32−–Zr4+–) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP. PMID:27754349

  15. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    PubMed

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃(2-)-Zr(4+)-) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  16. Fluorescence detection of adenosine triphosphate in an aqueous solution using a combination of copper(II) complexes.

    PubMed

    Kataev, Evgeny; Arnold, René; Rüffer, Tobias; Lang, Heinrich

    2012-08-06

    Fluorescent ligands have been designed to form ternary complexes with a Cu(II) cation and phosphates in a buffer solution at physiological pH 7.4. It has been shown that a combination of two different ligands and CuCl(2) allows one to achieve high adenosine triphosphate/adenosine diphosphate, adenosine 5'-monophosphate selectivity, and ratiometric fluorescence sensing, while separately each ligand complex does not have such properties.

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

    PubMed

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

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

  18. Use of extractable adenosine triphosphate to estimate the viable cell mass in dental plaque samples obtained from monkeys.

    PubMed Central

    Robrish, S A; Kemp, C W; Bowen, W H

    1978-01-01

    The viable cell mass in plaque samples obtained from monkeys was estimated by determining the concentration of extractable adenosine triphosphate (ATP), and total cell mass was estimated by measuring the protein content. The results were expressed in terms of the specific ATP and protein contents of Streptococcus sanguis. The viable counts estimated by these techniques were comparable to or exceeded viable counts obtained by other investigators using conventional bacteriological methods. PMID:417674

  19. Terbium ion-coordinated carbon dots for fluorescent aptasensing of adenosine 5'-triphosphate with unmodified gold nanoparticles.

    PubMed

    Xu, Mingdi; Gao, Zhuangqiang; Zhou, Qian; Lin, Youxiu; Lu, Minghua; Tang, Dianping

    2016-12-15

    This work reports on a novel time-resolved fluorescent aptasensing platform for the quantitative monitoring of adenosine 5'-triphosphate (ATP) by interaction of dispersive/agglomerate gold nanoparticles (AuNPs) with terbium ion-coordinated carbon dots (Tb-CDs). To construct such a fluorescent nanoprobe, Tb-CDs with high-efficient fluorescent intensity are first synthesized by the microwave method with terbium ions (Tb(3+)). The aptasensing system consists of ATP aptamer, AuNP and Tb-CD. The dispersive/agglomerate gold nanoparticles are acquired through the reaction of the aptamer with target ATP. Upon target ATP introduction, the aptamers bind with the analytes to form new aptamer-ATP complexes and coat on the surface of AuNPs to inhibit their aggregation in the high salt solution. In this case, the fluorescent signal of Tb-CDs is quenched by the dispersive AuNPs on the basis of the fluorescence resonance energy transfer (FRET). At the absence of target analyte, gold nanoparticles tend to aggregate in the high salt state even if the aptamers are present. Thus, the added Tb-CDs maintain their intrinsic fluorescent intensity. Experimental results indicated that the aptasensing system exhibited good fluorescent responses toward ATP in the dynamic range from 40nM to 4.0μM with a detection limit of 8.5nM at 3sblank criterion. The repeatability and intermediate precision is less than 9.5% at three concentrations including 0.04, 0.4 and 2.0μM ATP. The selectivity was acceptable toward guanosine 5'-triphosphate, uridine 5'-triphosphate and cytidine 5'-triphosphate. The methodology was applied to evaluate the blank human serum spiked with target ATP, and the recoveries (at 3 concentration levels) ranged between 97.0% and 103.7%. Importantly, this detection scheme is rapid, simple, cost-effective, and does not require extensive sample preparation or separation.

  20. 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.

  1. Antibodies to inositol 1,4,5-triphosphate receptor 1 in patients with cerebellar disease

    PubMed Central

    Fouka, Penelope; Alexopoulos, Harry; Chatzi, Ioanna; Dedos, Skarlatos G.; Samiotaki, Martina; Panayotou, George; Politis, Panagiotis; Tzioufas, Athanasios

    2016-01-01

    Objective: To describe newly identified autoantibodies associated with cerebellar disorders. Design/Methods: We first screened the sera of 15 patients with cerebellar ataxia, without any known associated autoantibodies, with immunocytochemistry on mouse brain. After characterization and validation of a newly identified antibody, 85 additional patients with suspected autoimmune cerebellar disease were screened using a cell-based assay. Results: Immunoglobulin G from one of the first 15 patients demonstrated a distinct staining pattern on Purkinje neurons. This autoantibody, as characterized further by immunoprecipitation and mass spectrometry, was binding inositol 1,4,5-triphosphate receptor 1 (IP3R1), an intracellular channel that mediates the release of Ca2+ from intracellular stores. Anti-IP3R1 specificity was then validated with a cell-based assay. On this basis, screening of 85 other patients with cerebellar disease revealed 2 additional IP3R1-positive patients. All 3 patients presented with cerebellar ataxia; the first was eventually diagnosed with primary progressive multiple sclerosis, the second had a homozygous CAG insertion at the gene TBP, and the third was thought to have a neurodegenerative disease. Conclusions: We independently identified an autoantibody against IP3R1, a protein highly expressed in Purkinje neurons, confirming an earlier report. Because a mouse knockout model for IP3R1 exhibits ataxia and epilepsy, this autoantibody may have a functional role. The heterogeneity of the antibody-positive patients suggests that this antibody may either have a direct involvement in disease pathogenesis or it is a surrogate marker secondary to cerebellar injury. Anti-IP3R1 antibodies should be further explored in various ataxic and epileptic syndromes as they may denote a marker of response to immunotherapies. PMID:27957507

  2. Optimization of adenosine 5'-triphosphate extraction for the measurement of acidogenic biomass utilizing whey wastewater.

    PubMed

    Lee, Changsoo; Kim, Jaai; Hwang, Seokhwan

    2006-08-01

    A set of experiments was carried out to maximize adenosine 5'-triphosphate (ATP) extraction efficiency from acidogenic culture using whey wastewater. ATP concentrations at different microbial concentrations increased linearly as microbial concentration decreased. More than 50% of ATP was extracted from the sample of 39 mg volatile suspended solids (VSS)/l compared to the sample of 2.8 g VSS/l. The ATP concentrations of the corresponding samples were 0.74+/-0.06 and 0.49+/-0.05 mg/l, respectively. For low VSS concentrations ranging from 39 to 92 mg/l, the extracted ATP concentration did not vary significantly at 0.73+/-0.01 mg ATP/l. Response surface methodology with a central composite in cube design for the experiments was used to locate the optimum for maximal ATP extraction with respect to boiling and bead beating treatments. The overall designed intervals were from 0 to 15 min and from 0 to 3 min for boiling and bead beating, respectively. The extracted ATP concentration ranged from 0.01 to 0.74 mg/l within the design boundary. The following is a partial cubic model where eta is the concentration of ATP and x ( k ) is the corresponding variable term (k=boiling time and bead beating time in order): eta=0.629+0.035x (1)-0.818x (2)-0.002x (1) x (2)-0.003x (1) (2) +0.254x (2) (2) +0.002x (1) (2) x (2). This model successfully approximates the response of ATP concentration with respect to the boiling- and bead beating-time. The condition for maximal ATP extraction was 5.6 min boiling without bead beating. The maximal ATP concentration using the model was 0.74 mg/l, which was identical to the experimental value at optimum condition for ATP extraction.

  3. Effects of caffeine on fractional flow reserve values measured using intravenous adenosine triphosphate.

    PubMed

    Nakayama, Masafumi; Chikamori, Taishiro; Uchiyama, Takashi; Kimura, Yo; Hijikata, Nobuhiro; Ito, Ryosuke; Yuhara, Mikio; Sato, Hideaki; Kobori, Yuichi; Yamashina, Akira

    2017-01-21

    We investigated the effects of caffeine intake on fractional flow reserve (FFR) values measured using intravenous adenosine triphosphate (ATP) before cardiac catheterization. Caffeine is a competitive antagonist for adenosine receptors; however, it is unclear whether this antagonism affects FFR values. Patients were evenly randomized into 2 groups preceding the FFR study. In the caffeine group (n = 15), participants were given coffee containing 222 mg of caffeine 2 h before the catheterization. In the non-caffeine group (n = 15), participants were instructed not to take any caffeine-containing drinks or foods for at least 12 h before the catheterization. FFR was performed in patients with more than intermediate coronary stenosis using the intravenous infusion of ATP at 140 μg/kg/min (normal dose) and 170 μg/kg/min (high dose), and the intracoronary infusion of papaverine. FFR was followed for 30 s after maximal hyperemia. In the non-caffeine group, the FFR values measured with ATP infusion were not significantly different from those measured with papaverine infusion. However, in the caffeine group, the FFR values were significantly higher after ATP infusion than after papaverine infusion (P = 0.002 and P = 0.007, at normal and high dose ATP vs. papaverine, respectively). FFR values with ATP infusion were significantly increased 30 s after maximal hyperemia (P = 0.001 and P < 0.001 for normal and high dose ATP, respectively). The stability of the FFR values using papaverine showed no significant difference between the 2 groups. Caffeine intake before the FFR study affected FFR values and their stability. These effects could not be reversed by an increased ATP dose.

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

    NASA Astrophysics Data System (ADS)

    Evans, M. G.; Marty, A.

    1986-06-01

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

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

    PubMed

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

    2014-09-01

    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.

  6. Antihyperlipidemic activity of adenosine triphosphate in rabbits fed a high-fat diet and hyperlipidemic patients.

    PubMed

    Zhang, Lianshan; Liang, Libin; Tong, Tong; Qin, Yuguo; Xu, Yanping; Tong, Xinglong

    2016-10-01

    Context Recently, adenosine triphosphate (ATP) was occasionally found to decrease the triglyceride (TG) levels in several hyperlipidemic patients in our clinical practice. Objective The study investigates the anti-hyperlipidemic effects of ATP in a high-fat fed rabbit model and hyperlipidemic patients. Materials and methods Twenty-four rabbits were randomly divided into three groups of eight animals each as follows: normal diet, high-fat diet and high-fat diet + ATP group. ATP supplementation (40 mg/day) was started at the 20th day and lasted for 10 days. Serum concentrations of total cholesterol (TC), TG, LDL-C, HDL-C were measured on the 20th day and 30th day. Heart, liver and aorta were subjected histopathological examination. Twenty outpatients diagnosed primary hyperlipidemia took ATP at a dose of 60 mg twice a day for 1 week. Results Feeding rabbits with a high-fat diet resulted in a significant elevation of lipid parameters including TC, TG, LDL-C, VLDL-C compared to the normal diet group (p < 0.01). ATP treatment significantly decreased serum TG level (p < 0.01), whilst other parameters remained statistically unaltered. Meanwhile, ATP significantly reduced the thickness of fat layer in cardiac epicardium (p < 0.05) and pathological gradation of ballooning degeneration in hepatocytes (p < 0.05). After taking ATP for 1 week, hyperlipidemia patients exhibited a significant decrease of TG (p < 0.01), but other lipid parameters had no significant change. Discussion and conclusion The study indicates that ATP selectively decreases serum TG levels in high-fat diet rabbits and hyperlipidemic patients. Therefore, ATP supplementation may provide an effective approach to control TG level.

  7. 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 οGads ° was estimated to only ⿿4 kJ/mol, the large value of Nmax led to significantly negative effective οGads values down to ⿿33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, ⿦).

  8. Thiamine triphosphate: a ubiquitous molecule in search of a physiological role.

    PubMed

    Bettendorff, Lucien; Lakaye, Bernard; Kohn, Gregory; Wins, Pierre

    2014-12-01

    Thiamine triphosphate (ThTP) was discovered over 60 years ago and it was long thought to be a specifically neuroactive compound. Its presence in most cell types, from bacteria to mammals, would suggest a more general role but this remains undefined. In contrast to thiamine diphosphate (ThDP), ThTP is not a coenzyme. In E. coli cells, ThTP is transiently produced in response to amino acid starvation, while in mammalian cells, it is constitutively produced at a low rate. Though it was long thought that ThTP was synthesized by a ThDP:ATP phosphotransferase, more recent studies indicate that it can be synthesized by two different enzymes: (1) adenylate kinase 1 in the cytosol and (2) FoF1-ATP synthase in brain mitochondria. Both mechanisms are conserved from bacteria to mammals. Thus ThTP synthesis does not seem to require a specific enzyme. In contrast, its hydrolysis is catalyzed, at least in mammalian tissues, by a very specific cytosolic thiamine triphosphatase (ThTPase), controlling the steady-state cellular concentration of ThTP. In some tissues where adenylate kinase activity is high and ThTPase is absent, ThTP accumulates, reaching ≥ 70% of total thiamine, with no obvious physiological consequences. In some animal tissues, ThTP was able to phosphorylate proteins, and activate a high-conductance anion channel in vitro. These observations raise the possibility that ThTP is part of a still uncharacterized cellular signaling pathway. On the other hand, its synthesis by a chemiosmotic mechanism in mitochondria and respiring bacteria might suggest a role in cellular energetics.

  9. 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

  10. 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

  11. Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation

    PubMed Central

    Gündüz, Dursun; Tanislav, Christian; Sedding, Daniel; Parahuleva, Mariana; Santoso, Sentot; Troidl, Christian; Hamm, Christian W.; Aslam, Muhammad

    2017-01-01

    Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention. PMID:28146050

  12. Monitoring of endoscope reprocessing with an adenosine triphosphate (ATP) bioluminescence method

    PubMed Central

    Parohl, Nina; Stiefenhöfer, Doris; Heiligtag, Sabine; Reuter, Henning; Dopadlik, Dana; Mosel, Frank; Gerken, Guido; Dechêne, Alexander; Heintschel von Heinegg, Evelyn; Jochum, Christoph; Buer, Jan; Popp, Walter

    2017-01-01

    Background: The arising challenges over endoscope reprocessing quality proposes to look for possibilities to measure and control the process of endoscope reprocessing. Aim: The goal of this study was to evaluate the feasibility of monitoring endoscope reprocessing with an adenosine triphosphate (ATP) based bioluminescence system. Methods: 60 samples of eight gastroscopes have been assessed from routine clinical use in a major university hospital in Germany. Endoscopes have been assessed with an ATP system and microbial cultures at different timepoints during the reprocessing. Findings: After the bedside flush the mean ATP level in relative light units (RLU) was 19,437 RLU, after the manual cleaning 667 RLU and after the automated endoscope reprocessor (AER) 227 RLU. After the manual cleaning the mean total viable count (TVC) per endoscope was 15.3 CFU/10 ml, and after the AER 5.7 CFU/10 ml. Our results show that there are reprocessing cycles which are not able to clean a patient used endoscope. Conclusion: Our data suggest that monitoring of flexible endoscope with ATP can identify a number of different influence factors, like the endoscope condition and the endoscopic procedure, or especially the quality of the bedside flush and manual cleaning before the AER. More process control is one option to identify and improve influence factors to finally increase the overall reprocessing quality, best of all by different methods. ATP measurement seems to be a valid technique that allows an immediate repeat of the manual cleaning if the ATP results after manual cleaning exceed the established cutoff of 200 RLU.

  13. Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

    PubMed

    Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

    2006-09-01

    A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

  14. 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.

  15. Effect of adenosine 5'-triphosphate infusions on the nutritional status and survival of preterminal cancer patients.

    PubMed

    Beijer, Sandra; Hupperets, Pierre S; van den Borne, Ben E; Eussen, Simone R; van Henten, Arjen M; van den Beuken-van Everdingen, Marieke; de Graeff, Alexander; Ambergen, Ton A; van den Brandt, Piet A; Dagnelie, Pieter C

    2009-08-01

    The aim of the study was to investigate the effect of intravenous infusions of adenosine 5'-triphosphate (ATP) on nutritional status and survival in preterminal cancer patients. Ninety-nine preterminal cancer patients (estimated life expectancy 1-6 months) with mixed tumor types were randomly allocated to receive either intravenous ATP weekly (8-10 h/week, maximum 50 microg/kg/min) for 8 weeks, or no ATP (control group). Nutritional status parameters were assessed until 8 weeks, and analyzed by repeated-measures analysis of covariance. Cox proportional hazards models were fitted to assess the effect of ATP on short-term (0-8 weeks) and long-term (0-6 months) survival. Fifty-one patients were randomized to ATP and 48 to the control group. Results showed a significant favorable effect of ATP on triceps skin fold thickness [between-group difference per 8 weeks 1.76 mm, 95% confidence interval (CI): 0.48-3.12 mm; P = 0.009] and on short-term survival [0-8 weeks hazard ratio (HR): 0.40, 95% CI: 0.17-0.95; P = 0.037]. In weight-stable patients and in lung cancer patients, long-term survival (0-6 months) was also significantly better in ATP-treated patients (weight-stable patients HR: 0.40, 95% CI: 0.19-0.83; P = 0.014; patients with lung cancer: HR: 0.35, 95% CI: 0.14-0.88; P = 0.025). In conclusion, in this population of preterminal cancer patients, ATP infusions, at the dose and schedule studied, had a favorable effect on triceps skin fold thickness and survival, especially in weight-stable patients and patients with lung cancer. Larger studies are warranted to confirm these findings and to further define the effect of ATP on tumor growth and survival.

  16. Suppression of the biosynthesis of guanosine triphosphate by protein synthesis inhibitors

    SciTech Connect

    Volkin, E.; Boling, M.E.; Jones, M.H.; Lee, W.H.; Pike, L.M.

    1980-10-10

    In a prior report it was observed that CTP synthesis and concomitant incorporation of CMP into RNA and dCMP into DNA were markedly reduced in cells cultured in the presence of cycloheximide and puromycin. Experiments described here with Novikoff hepatoma cells reveal that the purine biosynthetic pathway is similarly affected. When the cells are subjected to cycloheximide (30 or 60 ..mu..g/ml) or puromycin (100 ..mu..g/ml), there is a substantial reduction in the bioconversion of hypoxanthine, adenosine, and deoxyadenosine into guanylate compared to untreated cultures. Whereas synthesis (counts per min/nmol) of pool ATP was 70 to 100% of controls, that of pool GTP was 20 to 35% of controls. Incorporation of AMP into RNA was 40 to 60% of controls, but that of GMP was only 10 to 25% of controls. Incorporation of dAMP into DNA averaged 10% of controls, but that of dGMP was only 4% of controls. Synthesis of guanylates from formate by the de novo pathway was similarly reduced, but incorporation of guanosine, which enters via kinase action alone, was not disproportionately lowered. These results suggest that protein synthesis inhibitors cause a severely reduced availability of newly synthesized GTP and CTP as well as their deoxy counterparts, dGTP and dCTP, the proximal precursors for the synthesis of RNA and DNA. However, the nanomolar levels of all nucleoside triphosphates remain high, probably as a result of recycling of nucleic acid breakdown products. Thus, reduced synthesis of these compounds may restrict nucleic acid synthesis only of some sort of compartmentation leads to a limitation of these precursors at the site(s) of nucleic acid synthesis.

  17. Characterization of the nucleoside triphosphate phosphohydrolase I gene from the Choristoneura fumiferana entomopoxvirus.

    PubMed

    Li, X; Wallis, J L; Barrett, J W; Krell, P J; Arif, B M

    1998-07-01

    Poxviruses carry the enzyme, nucleoside triphosphate phosphohydrolase I (NPH I), required for early viral transcription in the cytoplasm of infected cells. The gene (nph I) encoding this enzyme from Choristoneura fumiferana entomopoxvirus (CfEPV) has been located in the viral genome, cloned and characterized. It has an open reading frame of 1941 nucleotides, potentially encoding a protein with a predicted molecular mass of 76.04 kDa and a pI of 8.83. It has a TAAATG motif where the trinucleotide ATG represents the translational start signal an AT-rich (88%) sequence and an early transcription termination signal (TTTTTAT) upstream of the ATG codon. Northern blot analysis of mRNA from infected larvae showed that a single 4.0 kb transcript which appeared late at day 20 post infection (p.i.) and its transcription continued till day 37 p.i.. Primer extension experiments suggested that the main transcripts started at 15 bases upstream of AUG codon. NPH I homologues have been found in the genomes of other entomopoxviruses and vertebrate poxviruses. Alignment of their amino acid sequences suggested three conserved domains, two of which are considered as ATP binding domains. The most similar homologue is from the closely related entomopoxvirus. Choristoneura biennis EPV (CbEPV) where 98.2% of nucleotide and 97.2% of amino acid identities are observed, respectively. A single nucleotide difference in CfEPV nph I was sufficient to distinguish it from CbEPV by PCR amplification and digestion with a restriction enzyme.

  18. Usefulness of adenosine triphosphate-atropine stress echocardiography for detecting coronary artery stenosis.

    PubMed

    Miyazono, Y; Kisanuki, A; Toyonaga, K; Matsushita, R; Otsuji, Y; Arima, S; Nakao, S; Tanaka, H

    1998-08-01

    There have been few studies on adenosine triphosphate (AT) stress echocardiography. The AT stress test may have fewer adverse effects than the adenosine stress test. The addition of atropine to AT echocardiography may enhance the sensitivity for detection of coronary artery disease (CAD). The purpose of this study was to determine the utility of AT-atropine echocardiography for detection of CAD. The group studied consisted of 112 patients with suspected CAD. Sixty-one patients did not have a history of prior myocardial infarction (group I) and 51 patients did (group II). AT was infused intravenously at 180 microg/kg/min for 14 minutes. Atropine (0.25 mg intravenously, repeated up to maximum total dose of 1 mg) was administered starting after 8 minutes of AT infusion. Ischemic response was defined as new or worsening wall motion abnormality occurring during the infusion. The sensitivity and specificity for detection of CAD were assessed using the representative echocardiograms during single AT infusion and AT-atropine infusion. Sixty-two patients had CAD. Fifty-eight patients (52%) developed minor side effects that resolved promptly. The rate-pressure product (10(3)/mm Hg beats/min) was significantly increased at 12 minutes of infusion (12.4+/-3.2) compared with that at baseline (9.1+/-2.3) and that at 6 minutes of infusion (9.4+/-2.1). The sensitivity for detection of CAD was 45% for AT echocardiography and 74% for AT-atropine echocardiography. The specificity was 94% for AT echocardiography and 90% for AT-atropine echocardiography. The sensitivity and specificity of AT-atropine echocardiography was 78% and 93%, respectively, in group I, and 70% and 86%, respectively, in group II. In conclusion, AT-atropine stress echocardiography seems to be well tolerated, safe, and useful for detection of CAD.

  19. Formycin triphosphate-terbium complex: a novel spectroscopic probe for phosphoryl transfer enzymes.

    PubMed

    Kirk, W R; Amzel, L M

    1987-12-18

    The conditions under which the fluorescent pyrazolopyrimidine nucleotide formycin A triphosphate (7-amino-3-(beta-D-(5'- tripolyphosphate)ribofuranosyl)pyrazolo[4,3-d]pyrimidine, FTP) forms a 1:1 complex in solution with Tb3+ have been characterized. The complex has a dissociation constant of approx. 10(-7) M. Within the complex, the luminescence of Tb3+ is dramatically sensitized by energy transfer from formycin. The value for 50% transfer efficiency, Förster's R0 (Förster, T. (1964) in Modern Quantum Chemistry (Sinanoglu, O., ed.), pp. 93-137, Academic Press, New York) was determined to be 3.34 +/- 0.4 A, and the effective distance between the donor and acceptor transition dipoles, R, in the complex was estimated to be 6.6 +/- 1.0 A. The quantum yield of Tb3+ in the complex is sensitive to the number of O-H oscillators bound to the Tb3+, which allows determination of the number of waters bound to it (approx. 4). Preliminary results show that the complex binds to the phosphoryl transfer enzyme hexokinase in the presence of the glucose analogs N-acetylglucosamine, frucose and xylose, which are not phosphorylated by the enzyme. The binding occurs with a loss of energy efficiency consistent with a new distance from the effective transition dipole of formycin to that of terbium of approx. 9.6 A. The FTP-terbium complex can be used as both a spectroscopic and an X-ray diffraction probe. Studies with this compound should be most valuable for correlating solution and crystallographic data.

  20. Emtricitabine-Triphosphate in Dried Blood Spots as a Marker of Recent Dosing.

    PubMed

    Castillo-Mancilla, Jose; Seifert, Sharon; Campbell, Kayla; Coleman, Stacey; McAllister, Kevin; Zheng, Jia-Hua; Gardner, Edward M; Liu, Albert; Glidden, David V; Grant, Robert; Hosek, Sybil; Wilson, Craig M; Bushman, Lane R; MaWhinney, Samantha; Anderson, Peter L

    2016-11-01

    New objective measures of antiretroviral adherence are needed. We determined if emtricitabine triphosphate (FTC-TP) in dried blood spots (DBS) can be used as a marker of recent dosing with tenofovir disoproxil fumarate-emtricitabine (TDF-FTC). The half-life of FTC-TP was estimated in DBS samples obtained from an intensive pharmacokinetic (PK) study of coformulated TDF-FTC in HIV-negative and HIV-infected participants. The concordance of quantifiable FTC-TP in DBS with tenofovir (TFV)/FTC in plasma was evaluated by utilizing paired plasma-DBS samples from participants enrolled in 2 large preexposure prophylaxis (PrEP) open-label trials. The time to FTC-TP nondetectability after TDF-FTC dosing was evaluated utilizing DBS from HIV-negative participants enrolled in a directly observed therapy study of variable adherence to TDF-FTC. The mean (95% confidence interval [CI]) terminal half-life of FTC-TP in the PK study was 35 (23 to 47) h. A total of 143/163 (88%) samples obtained 0 to 48 h post-TDF-FTC dose had quantifiable FTC-TP in DBS, compared with 2/93 (2%) and 0/87 (0%) obtained >48 and >96 h postdose. In 746 paired plasma-DBS samples from 445 participants enrolled in PrEP trials, when both TFV/FTC in plasma were below the limit of quantification, FTC-TP was as well in 98.9% of the samples, and when either TFV or FTC in plasma was quantifiable, FTC-TP was as well in 90.5% of the samples. The half-life of FTC-TP in DBS is short relative to that of TFV-diphosphate (TFV-DP), making it a surrogate for TFV-FTC detection in plasma. FTC-TP can be quantified in DBS simultaneously with TFV-DP, which quantifies cumulative adherence to TDF-FTC. (The clinical trials discussed in this article have been registered at ClinicalTrials.gov under identifiers NCT01040091, NCT02022657, NCT00458393, NCT01772823, and NCT02012621.).

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

    DOE PAGES

    Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; ...

    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

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

    PubMed

    Dumond, Julie B; 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

    2015-10-01

    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.

  3. 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.

  4. A putative nucleoside triphosphate-binding domain in the nonstructural protein of B19 parvovirus is required for cytotoxicity.

    PubMed Central

    Momoeda, M; Wong, S; Kawase, M; Young, N S; Kajigaya, S

    1994-01-01

    Cytotoxicity secondary to B19 parvovirus infection is due to expression of the viral nonstructural protein. Nonstructural proteins of many parvoviruses contain a well-conserved nucleoside triphosphate (NTP)-binding motif, which has been shown to be essential for a variety of protein functions. We show here that cytotoxicity of the B19 parvovirus nonstructural protein was abolished by single mutations of amino acids within the NTP-binding domain, especially within the A motif, implicating NTP-binding in virus-induced cell death. Images PMID:7966641

  5. Effects of adenosine triphosphate (ATP) on early recovery after total knee arthroplasty (TKA): a randomized, double-blind, controlled study.

    PubMed

    Long, Gong; Zhang, Guo Qiang

    2014-12-01

    Functional exercise after total knee arthroplasty (TKA) is necessary. However, it may be a difficult and painful process for the patient. Desirable methods of relieving the patient's pain are worth exploring. Oral supplement of adenosine triphosphate (ATP) is a potential option. In the present study, we decide to investigate whether short-term administration of ATP benefits patients undergoing TKA. A total of 244 subjects were randomized to receive 120mg ATP or placebo each day for 4weeks. Significant differences in quadriceps strength, pain scores at postoperative days 7, 14, 21, and 28 and total opioid consumption were detected. It follows that oral supplement of ATP could benefit patients recovering from TKA.

  6. 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.

  7. Formycin triphosphate as a probe for the ATP binding site involved in the activation of guanylate cyclase.

    PubMed

    Chang, C H; Yu, Z N; Song, D L

    1992-10-01

    Formycin A triphosphate (FTP), a fluorescent analog of ATP, slightly increased basal guanylate cyclase activity, but significantly potentiated guanylate cyclase activity stimulated by atrial natriuretic factor (ANF) in rat lung membranes. FTP potentiated ANF-stimulated guanylate cyclase activity with an EC50 at about 90 microM and inhibited ATP-stimulated guanylate cyclase activity with an IC50 at about 100 microM. These results indicate that FTP binds more tightly than ATP for the same binding site. Therefore, FTP would be an excellent tool for studying the ATP binding site.

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

    PubMed

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

    2015-05-07

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

  9. Enzymatic conversion of dihydroneopterin triphosphate to the pyrimidodiazepine intermediate involved in the biosynthesis of the drosopterins in Drosophila melanogaster.

    PubMed

    Wiederrecht, G J; Paton, D R; Brown, G M

    1984-02-25

    The compound 2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b]-[1,4]diazepine (pyrimidodiazepine or PDA, for short) is a precursor of the red eye pigments called the drosopterins in Drosophila melanogaster. The precursor of PDA is 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydrop teridine triphosphate (dihydroneopterin triphosphate or H2-NTP). The synthesis of of PDA from H2-NTP requires reduced glutathione, another thiol such as 2-mercaptoethanol, Mg2+, and at least three enzymes: one that is missing in the eye color mutant, sepia; one that is present only in limited quantities in the mutant, clot; and a third one that has been described as sepiapterin synthase A. The last enzyme is present only in relatively small quantities in the mutant, purple. Because PDA is two electrons more reduced than H2-NTP, it would appear that the reducing power needed for this transformation is probably supplied by glutathione. Oxidized glutathione cannot replace reduced glutathione in the system. The yield of PDA produced enzymatically from H2-NTP can be as high as 40% under optimal conditions.

  10. PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

    PubMed

    Flores-Juárez, Cyntia R; González-Jasso, Eva; Antaramian, Anaid; Pless, Reynaldo C

    2016-10-01

    GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

  11. Ab initio molecular dynamics studies on HIV-1 reverse transcriptase triphosphate binding site: implications for nucleoside-analog drug resistance.

    PubMed Central

    Alber, F.; Carloni, P.

    2000-01-01

    Quantum-chemical methods are used to shed light on the functional role of residues involved in the resistance of HIV-1 reverse transcriptase against nucleoside-analog drugs. Ab initio molecular dynamics simulations are carried out for models representing the adduct between the triphosphate substrate and the nucleoside binding site. The triphosphate is considered either deprotonated or protonated at the gamma-position. Although the protonated form already experiences large rearrangements in the ps time scale, the fully deprotonated state exhibits a previously unrecognized low-barrier hydrogen bond between Lys65 and gamma-phosphate. Absence of this interaction in Lys65-->Arg HIV-1 RT might play a prominent role in the resistance of this mutant for nucleoside analogs (Gu Z et al., 1994b, Antimicrob Agents Chemother 38:275-281; Zhang D et al., 1994, Antimicrob Agents Chemother 38:282-287). Water molecules present in the active site, not detected in the X-ray structure, form a complex H-bond network. Among these waters, one may be crucial for substrate recognition as it bridges Gln151 and Arg72 with the beta-phosphate. Absence of this stabilizing interaction in Gln151-->Met HIV-1 RT mutant may be a key factor for the known drug resistance of this mutant toward dideoxy-type drugs and AZT (Shirasaka T et al., 1995, Proc Natl Acad Sci USA 92:2398-2402: Iversen AK et al., 1996, J Virol 70:1086-1090). PMID:11206075

  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. Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets.

    PubMed Central

    Metz, S A; Meredith, M; Rabaglia, M E; Kowluru, A

    1993-01-01

    Recent studies suggest a permissive requirement for guanosine 5'-triphosphate (GTP) in insulin release, based on the use of GTP synthesis inhibitors (such as myocophenolic acid) acting at inosine monophosphate (IMP) dehydrogenase; herein, we examine the glucose dependency of GTP synthesis. Mycophenolic acid inhibited insulin secretion equally well after islet culture at 7.8 or 11.1 mM glucose (51% inhibition) but its effect was dramatically attenuated when provided at < or = 6.4 mM glucose (13% inhibition; P < 0.001). These observations were explicable by a stimulation of islet GTP synthesis derived from IMP since, at high glucose: (a) total GTP content was augmented; (b) a greater decrement in GTP (1.75 vs. 1.05 pmol/islet) was induced by mycophenolic acid; and (c) a smaller "pool" of residual GTP persisted after drug treatment. Glucose also accelerated GTP synthesis from exogenous guanine ("salvage" pathway) and increased content of a pyrimidine, uridine 5'-triphosphate (UTP), suggesting that glucose augments production of a common regulatory intermediate (probably 5-phosphoribosyl-1-pyrophosphate). Pathway-specific radiolabeling studies confirmed that glucose tripled both salvage and de novo synthesis of nucleotides. We conclude that steep changes in the biosynthesis of cytosolic pools of GTP occur at modest changes in glucose concentrations, a finding which may have relevance to the adaptive (patho) physiologic responses of islets to changes in ambient glucose levels. PMID:8349822

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

    SciTech Connect

    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 (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 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

  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. Incorporation of fludarabine and 1-beta-D-arabinofuranosylcytosine 5'-triphosphates by DNA polymerase alpha: affinity, interaction, and consequences.

    PubMed

    Gandhi, V; Huang, P; Chapman, A J; Chen, F; Plunkett, W

    1997-08-01

    Fludarabine and 1-beta-D-arabinofuranosylcytosine (ara-C) are effective nucleoside analogues for the treatment of leukemias when used as single agents or together. Recent trials of the fludarabine and ara-C therapy with or without growth factors suggested an improved clinical response by combining fludarabine and ara-C. The activity of these antimetabolites depends on their phosphorylation to the respective triphosphates, F-ara-ATP and ara-CTP. The principal mechanism through which these triphosphates cause cytotoxicity is incorporation into DNA and inhibition of further DNA synthesis. A model system of DNA primer extension on a defined template sequence was used to quantitate the consequences of incorporation of one or two analogues by human DNA polymerase alpha (pol alpha). The template (31-mer) was designed so that DNA pol alpha incorporated six deoxynucleotides (alternately G and T) on the 17-mer primer, followed by insertion of an A and then a C. The primer was then elongated with G and T to the full-length product. The apparent Kms of DNA pol alpha to incorporate these analogues (0. 053 and 0.077 microM, respectively) were similar to the Km for dCTP (0.037 microM) and dATP (0.044 microM), suggesting that the enzyme recognized these analogues and incorporated them efficiently on the growing DNA primer. The velocity of extension (Vmax) of these primers ranged between 0.53 and 0.77%/min when normal nucleotides were present. Once inserted at the 3'-terminus, F-ara-AMP or ara-CMP were poor substrates for extension. However, in reactions lacking dCTP and dATP and with high concentrations of ara-CTP, ara-CMP was inserted by pol alpha after incorporation of the F-ara-AMP residue. This tandem incorporation of the two analogues resulted in almost complete inhibition (99.3%) of further extension of the primer. In the presence of competing deoxynucleotides, each analogue resulted in a dose-dependent inhibition of DNA synthesis. When present together, inhibition of the

  17. Synthesis and application of charge-modified dye-labeled dideoxynucleoside-5'-triphosphates to 'direct-load' DNA sequencing.

    PubMed

    Finn, Patrick J; Sun, Lei; Nampalli, Satyam; Xiao, Haiguang; Nelson, John R; Mamone, J Anthony; Grossmann, Greg; Flick, Parke K; Fuller, Carl W; Kumar, Shiv

    2002-07-01

    A novel series of charge-modified, dye-labeled 2',3'-dideoxynucleoside-triphosphate terminators were synthesized and evaluated as reagents for DNA sequencing. These terminators possess an advantage over existing reagents in that no purification is required to remove unreacted nucleotide or associated breakdown products prior to electrophoretic separation of the sequencing fragments. This obviates the need for a time consuming post-reaction work up, allowing direct loading of DNA sequencing reaction mixtures onto a slab gel. Thermo Sequenase II DNA polymerase poorly incorporates the charge-modified terminators compared with regular dye-labeled terminators. However, extending the linker arm between dye and nucleotide and using a mutant form of a related DNA polymerase can in part mitigate the decrease in substrate efficiency. We also present evidence that these charge-modified terminators can relieve gel compression artefacts when used with dGTP in sequencing reactions.

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

    PubMed

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

    2015-09-22

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

  19. Visual and light scattering spectrometric method for the detection of melamine using uracil 5'-triphosphate sodium modified gold nanoparticles.

    PubMed

    Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

    2017-02-15

    A highly selective method was presented for colorimetric determination of melamine using uracil 5'-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  1. Competitive adsorption of Cu(II) and Cd(II) ions by chitosan crosslinked with epichlorohydrin-triphosphate.

    PubMed

    Laus, Rogério; de Fávere, Valfredo Tadeu

    2011-10-01

    In this study, chitosan (CTS) was crosslinked with both epichlorohydrin (ECH) and triphosphate (TPP), by covalent and ionic crosslinking reactions, respectively. The resulting adsorbent (CTS-ECH-TPP) was characterized by SEM, CHN, EDS, FT-IR and TGA analyses, and tested for metal adsorption. The adsorbent was used in batch experiments to evaluate the adsorption of Cu(II) and Cd(II) ions in single and binary metal solutions. In single metal solutions the maximum adsorption capacities for Cu(II) and Cd(II) ions, obtained by Langmuir model, were 130.72 and 83.75 mg g⁻¹, respectively. Adsorption isotherms for binary solutions showed that the presence of Cu(II) decreased Cd(II) adsorption due to a significant competition effect, that is, the adsorbent was selective towards Cu(II) rather than Cd(II).

  2. 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.

  3. Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5'-triphosphate.

    PubMed

    Guzmán Luna, Carolina; Costán-Longares, Ana; Lucena, Francisco; Jofre, Joan

    2009-10-01

    The feasibility of detecting somatic coliphages by phage infection of Escherichia coli WG5 and measurement of phage propagation by the lysis mediated release of the bacterial host adenylate kinase (AK) and adenosine 5'-triphosphate (ATP) detected by a bioluminescent signal was evaluated. After 2h of incubation, all cultures infected with reference bacteriophage phiX174 showed a significant increase in the bioluminescent signal, even with number of phages as low as less of 10 plaque forming units (PFU). Naturally occurring somatic coliphages ensured a significant bioluminescent signal after 3h of infection when >10 PFU were inoculated. These results indicate that an easy and reliable method to detect low numbers of coliphages in less than 3h is feasible.

  4. Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

    PubMed Central

    Halvas, Elias K.; Svarovskaia, Evguenia S.; Pathak, Vinay K.

    2000-01-01

    Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription. PMID:11044079

  5. 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.

  6. A novel aptasensor for the ultra-sensitive detection of adenosine triphosphate via aptamer/quantum dot based resonance energy transfer.

    PubMed

    Li, Zheng; Wang, Yijing; Liu, Ying; Zeng, Yongyi; Huang, Aimin; Peng, Niancai; Liu, Xiaolong; Liu, Jingfeng

    2013-09-07

    We designed a novel aptamer based biosensor (aptasensor) for ultrasensitive detection of adenosine triphosphate (ATP) through resonance energy transfer (RET). The ATP aptamer was modified with Cy3 at the 3' end, and a green quantum dot (525) was attached to the 5' end of its complementary sequence respectively. The ATP aptamer and its complementary sequence could assemble into a duplex structure in the absence of target ATP, and then decrease the distance between the quantum dot and Cy3 which could produce significant RET signal. Upon ATP binding, the ATP aptamer could dissociate with its complementary sequence and then increase the distance between the quantum dot and Cy3 which would significantly decrease the RET signal. Therefore, the ATP detection could be easily achieved through detection of the fluorescence intensity ratio between 525 nm and 560 nm. The results show that the emission fluorescence intensity ratio of 525/560 is linearly related to the logarithmic concentration of ATP. The linear range of this aptasensor is from 0.1 nM to 1 μM, and the detection limit is lower down to 0.01 nM. Excellent selectivity of this aptasensor for ATP has been demonstrated through the detection of thymidine triphosphate (TTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and adenosine diphosphate (ADP) respectively as control. The method we described here could easily detect ATP with excellent selectivity, linearity and sensitivity down to the nanomolar range, as well as avoid photobleaching.

  7. A visible light excitable fluorescent sensor for triphosphate/pyrophosphate based on a diZn2+ complex bearing an intramolecular charge transfer fluorophore.

    PubMed

    Su, Guangyu; Liu, Zhipeng; Xie, Zhijun; Qian, Fang; He, Weijiang; Guo, Zijian

    2009-10-14

    Triphosphate or pyrophosphate can be recognised by a diZn(2+) complex of bis(BPEA)-appended intramolecular charge transfer fluorophore 4-amino-7-aminosulfonyl-2,1,3-benzoxadiazole, displaying a 5-6 fold fluorescent enhancement at 576 nm.

  8. Adenosine 5' triphosphate evoked mobilization of intracellular calcium in central nervous system white matter of adult mouse optic nerve.

    PubMed

    James, G; Butt, A M

    1999-06-11

    Although it has been established that immature glial cells express functional purinergic receptors, the responsiveness of mature glial cells in vivo had not been elucidated. This question was addressed using fura-2 ratiometric measurements of [Ca2+]i in the adult mouse optic nerve, a central nervous system (CNS) white matter tract, taking advantage of the facts that (i), the optic nerve contains glial cells but not neurons and (ii), that fura-2 loads primarily astrocytes in isolated intact optic nerves. We show that adenosine 5' triphosphate (ATP) evoked an increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effect at 3 microm ATP, and with a rank order of agonist potency of ATP > ADP > alpha,beta-methyline-ATP > UDP > adenosine. The results indicate mainly P2Y and P2X components, consistent with the in vitro astroglial purinergic receptor profile. The in vivo response of mature glia to ATP may be important in their response to CNS damage.

  9. Chitosan-triphosphate nanoparticles for encapsulation of super-paramagnetic iron oxide as an MRI contrast agent.

    PubMed

    Sanjai, Chutimon; Kothan, Suchart; Gonil, Pattarapond; Saesoo, Somsak; Sajomsang, Warayuth

    2014-04-15

    Super-paramagnetic iron oxide nanoparticles (SPIONPs) were encapsulated at various concentrations within chitosan-triphosphate (SPIONPs-CS) nanoparticles using an ionotropic gelation method. The encapsulation of SPIONPs within CS nanoparticles enhanced their dispersion ability in aqueous solution, with all particles being lower than 130 nm in size and having highly positive surface charge. The SPIONPs-CS nanoparticles exhibited crystalline structure and super-paramagnetic behavior, as seen in non-encapsulated SPIONPs. The morphology of SPIONPs-CS nanoparticles showed that they almost spherical in shape. The effect of phantom environments (culture medium and 3% agar solution) on either T1 or T2 weighted MRI was investigated using a clinical 1.5T MRI scanner. The results revealed that 3% agar solution showed relaxation values higher than the culture medium, leading to a significant decrease in the MR image intensity. Our results demonstrated that the SPIONPs-CS nanoparticles can be applied as tissue-specific MRI contrast agents.

  10. 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

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

    PubMed

    Bhaumik, A; Shearin, A M; Delong, R; Wanekaya, A; Ghosh, K

    2014-08-14

    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.

  12. Adenosine triphosphate prevents serum deprivation-induced apoptosis in human mesenchymal stem cells via activation of the MAPK signaling pathways.

    PubMed

    Berlier, Jessica L; Rigutto, Sabrina; Dalla Valle, Antoine; Lechanteur, Jessica; Soyfoo, Muhammad S; Gangji, Valerie; Rasschaert, Joanne

    2015-01-01

    Human mesenchymal stem cells (hMSC) are multipotent cells derived from various sources including adipose and placental tissues as well as bone marrow. Owing to their regenerative and immunomodulatory properties, their use as a potential therapeutic tool is being extensively tested. However, one of the major hurdles in using cell-based therapy is the use of fetal bovine serum that can trigger immune responses, viral and prion diseases. The development of a culture medium devoid of serum while preserving cell viability is therefore a major challenge. In this study, we demonstrated that adenosine triphosphate (ATP) restrained serum deprivation-induced cell death in hMSC by preventing caspases 3/7 activation and modulating ERK1/2 and p38 MAPK signaling pathways. We also showed that serum deprivation conditions triggered dephosphorylation of the proapoptotic protein Bad leading to cell death. Adjunction of ATP restored the phosphorylation state of Bad. Furthermore, ATP significantly modulated the expression of proapoptopic and antiapoptotic genes, in favor of an antiapoptotic profile expression. Finally, we established that hMSC released a high amount of ATP in the extracellular medium when cultured in a serum-free medium. Collectively, our results demonstrate that ATP favors hMSC viability in serum deprivation conditions. Moreover, they shed light on the cardinal role of the MAPK pathways, ERK1/2 and p38 MAPK, in promoting hMSC survival.

  13. Nicking endonuclease-assisted recycling of target-aptamer complex for sensitive electrochemical detection of adenosine triphosphate.

    PubMed

    Hu, Tianxing; Wen, Wei; Zhang, Xiuhua; Wang, Shengfu

    2016-02-21

    An electrochemical biosensor was developed for the detection of adenosine triphosphate (ATP) based on target-induced conformation switching and nicking endonuclease (NEase)-assisted signal amplification. The electrochemical biosensor was constructed by base pairing and target recognition. After capture DNA hybridized with the gold electrode, a significant current of Methylene Blue (MB) was obtained by differential pulse voltammetry. In the presence of ATP, the hairpin DNA formed a G-quadruplex structure due to the specific recognition between hairpin DNA and ATP. Then the exposed part of the target-aptamer complex hybridized with the 3'-terminus of capture DNA to form a specific nicking site for Nb.BbvCI, which led to NEase-assisted target-aptamer complex recycling. The released target-aptamer complex hybridized with the remaining capture DNA. Nb.BbvCI-assisted target-aptamer complex recycling caused the continuous cleavage of capture DNA with MB at its 5'-terminus, resulting in release of a certain amount of DNA fragment labeled with MB. Then the current value decreased significantly. The reduced current showed a linear range from 10 nM to 1 μM with a limit of detection as low as 3.4 nM. Furthermore, the proposed strategy can be used for the detection of similar substances.

  14. Adenosine triphosphate-competitive mTOR inhibitors: a new class of immunosuppressive agents that inhibit allograft rejection.

    PubMed

    Rosborough, B R; Raïch-Regué, D; Liu, Q; Venkataramanan, R; Turnquist, H R; Thomson, A W

    2014-09-01

    The mechanistic/mammalian target of rapamycin (mTOR) is inhibited clinically to suppress T cell function and prevent allograft rejection. mTOR is the kinase subunit of two mTOR-containing complexes, mTOR complex (mTORC) 1 and 2. Although mTORC1 is inhibited by the macrolide immunosuppressant rapamycin (RAPA), its efficacy may be limited by its inability to block mTORC1 completely and its limited effect on mTORC2. Adenosine triphosphate (ATP)-competitive mTOR inhibitors are an emerging class of mTOR inhibitors that compete with ATP at the mTOR active site and inhibit any mTOR-containing complex. Since this class of compounds has not been investigated for their immunosuppressive potential, our goal was to determine the influence of a prototypic ATP-competitive mTOR inhibitor on allograft survival. AZD8055 proved to be a potent suppressor of T cell proliferation. Moreover, a short, 10-day course of the agent successfully prolonged murine MHC-mismatched, vascularized heart transplant survival. This therapeutic effect was associated with increased graft-infiltrating regulatory T cells and reduced CD4(+) and CD8(+) T cell interferon-γ production. These studies establish for the first time, that ATP-competitive mTOR inhibition can prolong organ allograft survival and warrant further investigation of this next generation mTOR inhibitors.

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

    PubMed

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

    2013-10-01

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

  16. 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

  17. Force engages vinculin and promotes tumor progression by enhancing PI3-kinase activation of phosphatidylinositol (3,4,5)-triphosphate

    PubMed Central

    Rubashkin, MG; Cassereau, L; Bainer, R; DuFort, CC; Yui, Y; Ou, G; Paszek, MJ; Davidson, MW; Chen, YY; Weaver, VM

    2014-01-01

    Extracellular matrix stiffness induces focal adhesion assembly to drive malignant transformation and tumor metastasis. Nevertheless, how force alters focal adhesions to promote tumor progression remains unclear. Here, we explored the role of the focal adhesion protein vinculin, a force-activated mechano-transducer, in mammary epithelial tissue transformation and invasion. We found that extracellular matrix stiffness stabilizes the assembly of a vinculin-talin-actin scaffolding complex that facilitates PI3-kinase mediated phosphatidylinositol (3,4,5)-triphosphate phosphorylation. Using defined two and three dimensional matrices, a mouse model of mammary tumorigenesis with vinculin mutants and a novel super resolution imaging approach, we established that ECM stiffness, per se, promotes the malignant progression of a mammary epithelium by activating and stabilizing vinculin and enhancing Akt signaling at focal adhesions. Our studies also revealed that vinculin strongly co-localizes with activated Akt at the invasive border of human breast tumors, where the ECM is stiffest and we detected elevated mechano-signaling. Thus, extracellular matrix stiffness could induce tumor progression by promoting the assembly of signaling scaffolds; a conclusion underscored by the significant association we observed between highly expressed focal adhesion plaque proteins and malignant transformation across multiple types of solid cancer. PMID:25183785

  18. 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

  19. Electrochemiluminescence of blue-luminescent graphene quantum dots and its application in ultrasensitive aptasensor for adenosine triphosphate detection.

    PubMed

    Lu, Juanjuan; Yan, Mei; Ge, Lei; Ge, Shenguang; Wang, Shaowei; Yan, Jixian; Yu, Jinghua

    2013-09-15

    A simple approach based on exfoliating and disintegrating treatments for graphite oxide, followed by hydrothermal synthesis, was developed to prepare water-soluble graphene quantum dots (GQDs). The as-prepared GQDs exhibited bright blue emission under ultraviolet irradiation (∼365nm), and showed an excitation-independent photoluminescence feature. More importantly, a newly anodic electrochemiluminescence (ECL) was observed from the water-soluble GQDs with H2O2 as coreactant for the first time, and the ECL induced a strong light emission at a low potential (ca. 0.4V vs. Ag/AgCl). The ECL mechanism is investigated in detail. Employing SiO2 nanospheres as signal carrier, a novel SiO2/GQDs ECL signal amplification labels were synthesized based on which a ultrasensitive ECL aptamer sensor was proposed. Under the optimized experimental conditions, the proposed ECL aptamer sensor exhibited excellent analytical performance for adenosine triphosphate (ATP) determination, ranging from 5.0×10(-12) to 5.0×10(-9)molL(-1) with the detection limit of 1.5×10(-12)molL(-1). Due to the low cytotoxicity and excellent biocompatibility, GQDs are demonstrated to be an eco-friendly material as well as excellent ECL labeling agents for biosensor.

  20. Growth hormone deficiency in a dopa-responsive dystonia patient with a novel mutation of guanosine triphosphate cyclohydrolase 1 gene.

    PubMed

    Lin, Yu; Wang, Dan-Ni; Chen, Wan-Jin; Lin, Xiang; Lin, Min-Ting; Wang, Ning

    2015-05-01

    Dopa-responsive dystonia is a rare hereditary movement disorder caused by mutations in the guanosine triphosphate cyclohydrolase 1 (GCH1) gene. This disease typically manifests in dystonia, with marked diurnal fluctuation and a dramatic response to levodopa. However, growth retardation in dopa-responsive dystonia has rarely been reported, and the etiology of short stature is not clarified. Here, we report a 14-year-old patient with extremities dystonia and short stature. Treatment with levodopa relieved his symptoms and resulted in a height increase. We also investigated the mutation in GCH1 and the etiology of short stature in this case. Sequence analysis of GCH1 revealed a novel mutation (c.695G>T). Laboratory examinations and imaging confirmed the diagnosis of growth hormone deficiency. We conclude that our case reveals a rare feature for dopa-responsive dystonia and suggests a possible pathogenic link between growth hormone deficiency and dopa-responsive dystonia. We recommend levodopa as the first choice for treating dopa-responsive dystonia in children with growth hormone deficiency.

  1. Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase

    SciTech Connect

    Pandey, V.N.; Modak, M.J.

    1989-01-15

    Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with (/sup 32/P)ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of (/sup 32/P)ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identical with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide. The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.

  2. Controlled injection of a liquid into ultra-high vacuum: Submonolayers of adenosine triphosphate deposited on Cu(110)

    NASA Astrophysics Data System (ADS)

    Sobrado, J. M.; Martín-Gago, J. A.

    2016-10-01

    We have combined a fast-valve device with vacuum technology for implementing a new method that allows introducing liquid solutions in an ultra-high vacuum chamber in the form of very small droplets. This technical development allows the easy deposition of (bio) organic molecules or small nanoparticles on a surface in a fully in-situ process, avoiding possible contamination due to the handle of the material. Moreover, our experimental set-up is suitable for any liquid and does not require any voltage application as in electrospray. We can easily change the operating regime from liquid droplet injection to the formation of a highly dispersive jet of micro-droplets by exclusively adjusting external parameters. Due to the nature of the injection process, the operational protocol makes possible the deposition of delicate molecular species that cannot be thermally sublimated. In particular, we have used this system to study the deposition of adenosine triphosphate on Cu(110). The structure of the layer was analyzed by X-ray photoemission spectroscopy and the evolution of the signal from the deposited molecule with the number of injections indicates that the molecular coverage can be controlled with submonolayer precision.

  3. Peroxynitrite-dependent zinc release and inactivation of guanosine 5'-triphosphate cyclohydrolase 1 instigate its ubiquitination in diabetes.

    PubMed

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

    2013-12-01

    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.

  4. 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.

  5. Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

    PubMed

    Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

    2015-01-20

    Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration.

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

    PubMed Central

    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

  7. Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography.

    PubMed

    Zhou, Ling; Xue, XiaoFeng; Zhou, JinHui; Li, Yi; Zhao, Jing; Wu, LiMing

    2012-09-12

    To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.

  8. 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

  9. Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

    PubMed

    Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

    2015-03-03

    A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

  10. The role of microorganisms in the degradation of adenosine triphosphate (ATP) in chill-stored common carp (Cyprinus carpio) fillets.

    PubMed

    Li, Dapeng; Zhang, Longteng; Song, Sijia; Wang, Zhiying; Kong, Chunli; Luo, Yongkang

    2017-06-01

    Biochemical and microbial changes after harvest strongly affect the final quality and shelf life of fish and fish products. In this study, the role of microbes in the degradation of adenosine triphosphate (ATP), and the origin of adenosine monophosphate deaminase (AMPD) and acid phosphatase (ACP) in common carp fillets during different stages of chilled storage (at 4°C) were investigated. The content of ATP, ADP, AMP, IMP, HxR, and Hx, the activity of AMPD and ACP, and the total count of viable, Aeromonas, Pseudomonas, H2S-producing bacteria, and lactic acid bacteria were examined. Results indicated that the population of microbial communities in control samples increased with storage time, and Pseudomonas peaked on the 10th day of storage. Changes in AMPD activity were less related to the abundance of microbes during the entire storage period. However, ACP was derived from both fish muscle and microbial secretion during the middle and late stages of storage. Degradation of ATP to IMP was not affected by spoilage bacteria, but the hydrolysis of IMP, and the transformation of HxR to Hx was affected considerably by the spoilage bacteria.

  11. Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates.

    PubMed

    van der Vliet, P C; Kwant, M M

    1981-04-28

    In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.

  12. A sensor for adenosine triphosphate fabricated by laser-induced forward transfer of luciferase onto a poly(dimethylsiloxane) microchip

    NASA Astrophysics Data System (ADS)

    Tsuboi, Yasuyuki; Furuhata, Yosuke; Kitamura, Noboru

    2007-08-01

    Laser-induced forward transfer (LIFT) of the enzyme luciferase was explored as a potential technique to be used in the fabrication of a microchip adenosine triphosphate (ATP) sensor. Poly(dimethylsiloxane) (PDMS) was selected as the substrate for deposition of the luciferase. In comparison with other solid substrates, such as glass and polystyrene, it was found that the flexibility of PDMS made it a superior substrate for the immobilization of micro-spots of luciferase. LIFT of luciferase onto a PDMS substrate using a 355 nm laser was successfully carried out, while the bioactivity of the enzyme was maintained. Yellow luminescence ascribed to luciferase was observed from a transferred spot on the PDMS chip from the enzymatic reaction between luciferin and ATP. A microchip ATP sensor was also fabricated by attaching a small photodiode to the PDMS chip. On the basis of the fabricated microchip, the Michaelis-Menten relation between the luminescence intensity from the spot, and the ATP concentration was confirmed. The potential for fabricating biosensors using a combination of the LIFT technique with a PDMS substrate was shown to be very good.

  13. 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.

  14. Action of angiotensin II, 5-hydroxytryptamine and adenosine triphosphate on ionic currents in single ear artery cells of the rabbit.

    PubMed

    Hughes, A D; Bolton, T B

    1995-10-01

    1. Angiotensin II, 5-hydroxytryptamine (5-HT) and adenosine triphosphate (ATP) evoked a transient inward current in isolated single car artery cells of rabbit held at -60 mV by whole cell voltage clamp in physiological saline using a KCL-containing pipette solution. Under these conditions agonist did not activate a calcium-dependent potassium current. 2. Responses to each agonist were transient and desensitized rapidly. Inward current at -60 mV holding potential was not abolished by blockade of voltage-dependent calcium channels or by buffering intracellular calcium with BAPTA, a calcium chelator, or following depletion of intracellular calcium stores with ryanodine. 3. The shape of the current-voltage relationships and the reversal potentials of the current induced by angiotensin II, 5-HT and ATP were similar under a variety of ionic conditions. Agonist-induced current was unaffected by replacing intracellular chloride with citrate ions or by replacing intracellular sodium with caesium or extracellular sodium with barium or calcium. Replacement of extracellular sodium with Tris shifted the reversal potential in all cases by around 30 mV negatively. 4. These data suggest that angiotensin II, 5-HT and ATP activate similar cationic conductances which are relatively non-selective allowing mono- and divalent cations to cross the smooth muscle cell membrane. These channels may allow the influx of calcium under physiological conditions.

  15. 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

  16. Efficient incorporation of positively charged 2', 3'-dideoxynucleoside-5'-triphosphates by DNA polymerases and their application in 'direct-load' DNA sequencing.

    PubMed

    Finn, Patrick J; Bull, Matthew G; Xiao, Haiguang; Phillips, Paula D; Nelson, John R; Grossmann, Greg; Nampalli, Satyam; McArdle, Bernard F; Mamone, J Anthony; Flick, Parke K; Fuller, Carl W; Kumar, Shiv

    2003-08-15

    A series of charge-modified, dye-labeled 2', 3'-dideoxynucleoside-5'-triphosphates have been synthesized and evaluated as reagents for dye-terminator DNA sequencing. Unlike the commonly used dye-labeled terminators, these terminators possess a net positive charge and migrate in the opposite direction to dye-labeled Sanger fragments during electrophoresis. Post-sequencing reaction purification is not required to remove unreacted nucleotide or associated breakdown products prior to electrophoresis. Thus, DNA sequencing reaction mixtures can be loaded directly onto a separating medium such as a sequencing gel. The charge-modified nucleotides have also been shown to be more efficiently incorporated by a number of DNA polymerases than regular dye-labeled dideoxynucleotide terminators or indeed normal dideoxynucleoside-5'-triphosphates.

  17. Appearance of adenosine triphosphate in the coronary sinus effluent from isolated working rat heart in response to hypoxia.

    PubMed Central

    Clemens, M G; Forrester, T

    1981-01-01

    1. A working rat heart preparation was used to study the release of adenosine-5'-triphosphate (ATP) into the coronary sinus effluent in response to hypoxia. 2. The left ventricle was set to pump against an hydrostatic pressure of 65 cm water; the left atrial filling pressure was kept constant at 10 cm water. The power output of the heart at these pressures was estimated to be approximately one half of the maximum power development. 3. Samples for ATP assay were collected (a) 30 sec before onset of hypoxia, (b) 60-90 sec after onset of hypoxia, (c) 5 min after restoration of oxygenated buffer solution. Respective concentrations of ATP were (nM +/- S.E.) 0.63 (+/- 0.18), 4.70 (+/- 0.39) and 0.63 (+/- 0.06). The total amounts of ATP detected were (p-mole/min) 5.9 (+/- 0.9), 46.1 (+/- 6.0) and 5.5 (+/- 1.2) respectively. 4. Viability of the hearts was judged to be satisfactory on the following grounds. Alterations in left atrial filling pressure produced typical Frank-Starling responses of the left ventricle. Oxygen extraction from the perfusate increased in response to increased workload. Coronary blood flow increased immediately upon introduction of hypoxic conditions and mechanical recovery from hypoxia was always complete within 5 min of restoring oxygen. 5. In view of the marked extracellular ATPase activity it is concluded that significant vasodilatory concentrations of ATP are released into the myocardial extracellular space in response to hypoxia. A scheme is proposed describing the possible role of adenine nucleotides in the local control of myocardial blood flow. PMID:7264990

  18. Intrapulmonary arteries respond to serotonin and adenosine triphosphate in broiler chickens susceptible to idiopathic pulmonary arterial hypertension.

    PubMed

    Kluess, H A; Stafford, J; Evanson, K W; Stone, A J; Worley, J; Wideman, R F

    2012-06-01

    This study examined factors contributing to increased vascular resistance and plexiform lesion formation in broiler chickens susceptible to idiopathic pulmonary arterial hypertension (IPAH). A diet supplemented with excess tryptophan (high-Trp diet), the precursor for serotonin, was used to accelerate the development of IPAH. Broilers fed the high-Trp diet had higher pulmonary arterial pressures than broilers fed the control diet, and plexiform lesion incidences tended to be higher (P = 0.11) in the high-Trp group than in the control group at 30 d of age. The intrapulmonary arteries were assessed for vasoconstriction in response to serotonin and adenosine triphosphate (ATP) and for activities of key metabolic enzymes for serotonin and ATP. The pulmonary artery (defined as the first major branch of the pulmonary artery inside the lung) and the primary pulmonary arterial rami (defined as the second major branch of the pulmonary artery inside the lung) both exhibited vasoconstriction in response to serotonin and ATP. This is the first study to demonstrate purinergic-mediated vasoconstriction in intrapulmonary arteries from broilers. Arteriole responsiveness did not differ between broilers fed the control diet or the high-Trp diet. Therefore, the high-Trp diet enhanced the development of IPAH but did not affect the artery's sensitivity to serotonin or ATP. Monoamine oxidase activity, responsible for the breakdown of serotonin, was severely impaired in pulmonary arteries from broilers in the high-Trp group. Accordingly, serotonin may persist longer and elicit an amplified response in broilers fed the high-Trp diet.

  19. A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

    PubMed

    Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

    2014-08-07

    A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

  20. Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5' to 3' Translocase in Transcription Termination of Vaccinia Early Genes.

    PubMed

    Hindman, Ryan; Gollnick, Paul

    2016-07-08

    Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5' to 3' translocase on single-stranded DNA.

  1. 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

  2. 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.

  3. Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

    PubMed

    Karner, Susanne; Shi, Shaojun; Fischer, Christine; Schaeffeler, Elke; Neurath, Markus F; Herrlinger, Klaus R; Hofmann, Ute; Schwab, Matthias

    2010-04-01

    6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

  4. Self-association of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) and promotion by metal ions.

    PubMed

    Scheller, K H; Sigel, H

    1986-05-15

    The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP4-) has been measured in D2O at 27 degrees C to elucidate the self-association. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K = 1.9 +/- 0.2 M-1, is only slightly larger than the value for ATP4-, K = 1.3 +/- 0.2 M-1. The self-stacking tendency of epsilon-ATP4- is promoted by a factor of about 4 by (1:1) coordination of Mg2+ to the phosphate moieties, which probably links these together and also neutralizes part of the negative charge; Zn2+ is only about half as effective as Mg2+ in promoting the self-association. This result contrasts with the self-stacking properties of Mg(ATP)2- and Zn(ATP)2-, Zn2+ being considerably more effective in a 1:1 ATP system. It is assumed that due to the enhanced affinity of the N-6/N-7 site of the epsilon-adenine moiety towards Zn2+ repulsion of the bases occurs resulting thus in a lower stacking tendency; in addition, the simple isodesmic model is no longer applicable to the Zn(epsilon-ATP)2- system: to explain the experimental data, the formation of an intermolecular metal ion bridge in the dimeric stacks is proposed. The experimental conditions required for studies of the properties of monomeric epsilon-ATP systems are described. Care should be exercised in employing epsilon-ATP as a probe for ATP.

  5. Borrelia burgdorferi rel Is Responsible for Generation of Guanosine-3′-Diphosphate-5′-Triphosphate and Growth Control

    PubMed Central

    Bugrysheva, Julia V.; Bryksin, Anton V.; Godfrey, Henry P.; Cabello, Felipe C.

    2005-01-01

    The global transcriptional regulator (p)ppGpp (guanosine-3′-diphosphate-5′-triphosphate and guanosine-3′,5′-bisphosphate, collectively) produced by the relA and spoT genes in Escherichia coli allows bacteria to adapt to different environmental stresses. The genome of Borrelia burgdorferi encodes a single chromosomal rel gene (BB0198) (B. burgdorferi rel [relBbu]) homologous to relA and spoT of E. coli. Its role in (p)ppGpp synthesis, bacterial growth, and modulation of gene expression has not been studied in detail. We constructed a relBbu deletion mutant in an infectious B. burgdorferi 297 strain and isolated an extrachromosomally complemented derivative of this mutant. The mutant did not synthesize relBbu mRNA, RelBbu protein, or (p)ppGpp. This synthesis was restored in the complemented derivative, confirming that relBbu is necessary and sufficient for (p)ppGpp synthesis and degradation in B. burgdorferi. The relBbu mutant grew well during log phase in complete BSK-H but reached lower cell concentrations in the stationary phase than the wild-type parent, suggesting that (p)ppGpp may be an important factor in the ability of B. burgdorferi to adapt to stationary phase. Deletion of relBbu did not eliminate the temperature-elicited OspC shift, nor did it alter bmp gene expression or B. burgdorferi antibiotic susceptibility. Although deletion of relBbu eliminated B. burgdorferi virulence for mice, which was not restored by complementation, we suggest that relBbu-dependent accumulation of (p)ppGpp may be important for in vivo survival of this pathogen. PMID:16041012

  6. Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

    PubMed

    Herman, Brian D; Schinazi, Raymond F; Zhang, Hong-wang; Nettles, James H; Stanton, Richard; Detorio, Mervi; Obikhod, Aleksandr; Pradère, Ugo; Coats, Steven J; Mellors, John W; Sluis-Cremer, Nicolas

    2012-01-01

    β-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

  7. Rapid and direct estimation of active biomass on granular activated carbon through adenosine tri-phosphate (ATP) determination.

    PubMed

    Velten, Silvana; Hammes, Frederik; Boller, Markus; Egli, Thomas

    2007-05-01

    Granular activated carbon (GAC) filtration is used during drinking water treatment for the removal of micropollutants such as taste and odour compounds, halogenated hydrocarbons, pesticides and pharmaceuticals. In addition, the active microbial biomass established on GAC is responsible for the removal of biodegradable dissolved organic carbon compounds present in water or formed during oxidation (e.g., ozonation and chlorination) processes. In order to conduct correct kinetic evaluations of DOC removal during drinking water treatment, and to assess the state and performance of full-scale GAC filter installations, an accurate and sensitive method for active biomass determination on GAC is required. We have developed a straight-forward method based on direct measurement of the total adenosine tri-phosphate (ATP) content of a GAC sample and other support media. In this method, we have combined flow-cytometric absolute cell counting and ATP analysis to derive case-specific ATP/cell conversion values. In this study, we present the detailed standardisation of the ATP method. An uncertainty assessment has shown that heterogeneous colonisation of the GAC particles makes the largest contribution to the combined standard uncertainty of the method. The method was applied for the investigation of biofilm formation during the start-up period of a GAC pilot-scale plant treating Lake Zurich water. A rapid increase in the biomass of up to 1.1 x 10(10)cells/g GAC dry weight (DW) within the first 33 days was observed, followed by a slight decrease to an average steady-state concentration of 7.9 x 10(9)cells/g GAC DW. It was shown that the method can be used to determine the biomass attached to the GAC for both stable and developing biofilms.

  8. 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).

  9. Nitrite-induced methemoglobinaemia affects blood ionized and total magnesium level by hydrolysis of plasma adenosine triphosphate in rat.

    PubMed

    Rahman, Md Mizanur; Kim, Shang-Jin; Kim, Gi-Beum; Hong, Chul-Un; Lee, Young-Up; Kim, Sung-Zoo; Kim, Jin-Shang; Kang, Hyung-Sub

    2009-11-01

    The objective of this study was to evaluate the effects of sodium nitrite (NaNO(2))-induced methemoglobinaemia on plasma ATP (adenosine triphosphate) and corresponding changes of blood-ionized magnesium (iMg(2+)) as well as total magnesium (tMg(2+)) in a time-dependent manner. This study was performed on male Sprague-Dawley rats to which NaNO(2) was injected (10 mg/kg i.p.) to induce methemoglobinaemia. Methemoglobin (MetHb) in blood was measured before (0 min.) and after 10, 30, 60 and 120 min. of NaNO(2) injection. At respective time points, the tMg(2+), blood ions and gases were measured by atomic absorption spectrometry and ion selective electrode, respectively. Haematological parameters were checked by automatic blood cell count, and blood films were observed under light microscope. Plasma ATP was measured by bioluminescence assay using a luminometer, and plasma proteins were measured by an automatic analyser. Blood cell count (RBC, WBC and platelet), haematocrit, and haemoglobin were found to be decreased with the advancement of MetHb concentration. With the gradual increase of MetHb concentration, the plasma ATP decreased and blood iMg(2+) and plasma tMg(2+) increased significantly as time passed by in comparison with the pre-drug values. A significant decrease of the ratio of ionized calcium to iMg(2+), Na(+) and increase of K(+) was observed. In conclusion, NaNO(2)-induced methemoglobinaemia is a cause of hydrolysis of plasma ATP which is responsible for the increase of blood iMg(2+) and plasma tMg(2+) in rats.

  10. Effects of cyclooxygenase-2 inhibitor and adenosine triphosphate-sensitive potassium channel opener in syngeneic mouse islet transplantation.

    PubMed

    Juang, J-H; Kuo, C-H

    2010-12-01

    In the initial days after transplantation, islet grafts may be attacked by cytokines via cyclooxygenase-2 (COX-2), producing primary nonfunction. In addition, chronic overstimulation of β-cells may impair insulin secretion. To enhance the function of transplanted islets, the present study investigated the effects of rofecoxib, a COX-2 inhibitor, and NN414 (6-chloro-3-[1-methylcyclopropyl]amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), an adenosine triphosphate-sensitive potassium channel opener, on islet transplantation. Male inbred C57BL/6 mice were used as donors and recipients. One hundred fifty islets were isolated via collagenase digestion and density gradient, and syngeneically transplanted under the kidney capsule in mice with streptozotocin-induced diabetes. Recipients were treated with or without rofecoxib, 10 mg/kg/d orally, or with or without NN414, 3 mg/kg/d orally, for 4 weeks. After transplantation, recipient body weight, blood glucose concentration, and intraperitoneal glucose tolerance were measured. The grafted kidney was extracted for determination of insulin content at 4 weeks. In the rofecoxib-treated and NN414-treated groups and both control groups, body weight remained stable, and the blood glucose concentration decreased progressively. However, at 4 weeks after transplantation in the groups treated or not treated with rofecoxib or NN414, no significant difference was observed in recipient body weight, blood glucose concentration, and glucose tolerance or in insulin content of the graft. These data indicate that posttransplantation treatment with rofecoxib or NN414 has no beneficial effect on transplantation outcome in diabetic mouse recipients engrafted with a marginal islet mass.

  11. 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.

  12. Creation of reduced graphene oxide based field effect transistors and their utilization in the detection and discrimination of nucleoside triphosphates.

    PubMed

    Yu, Chunmeng; Chang, Xingmao; Liu, Jing; Ding, Liping; Peng, Junxia; Fang, Yu

    2015-05-27

    Two low-cost, micropatterned, solution-gated field effect transistors (modified FET and unmodified FET) based on reduced graphene oxide (RGO) were developed and used for detection and discrimination of nucleoside triphosphates (NTPs). The modified FET was realized by simple deposition of a positively charged bis-pyrenyl derivative, py-diIM-py, onto the conducting RGO strips of the unmodified FET. The electrical properties and sensing behaviors of the as-prepared devices were studied comprehensively. Electrical transfer property tests revealed that both of the two FETs exhibit V-shaped ambipolar field effect behavior from p-type region to n-type region. Sensing performance studies demonstrated that modification of the native FET with py-diIM-py improves its sensing ability to NTPs-GTP and ATP in particular. The detection limit of GTP and ATP was as low as 400 nM, which is the lowest value for graphene-based electronic sensors reported so far. Furthermore, based on the cross-reactive responses of the two devices to NTPs, NTPs can be conveniently distinguished via combining use of the two devices. The enhancement of the modifier (py-diIM-py) to the sensing performance of the FET is tentatively attributed to its possible mediation role in sticking onto RGO strips and accumulating analytes by electrostatic association with the relevant species. Because they are sensitive and fast in response, simple and low-cost in preparation, and possibly useful in sensor-array fabrication, the developed sensors show great potential in real-life application.

  13. 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

  14. 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

  15. Beneficial effect of extracellular adenosine 5'-triphosphate treatment on the Indochinese leopard (Panthera pardus delacouri) sperm quality after cryopreservation.

    PubMed

    Thuwanut, P; Tipkantha, W; Siriaroonrat, B; Comizzoli, P; Chatdarong, K

    2016-11-22

    The Indochinese leopard (Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization (IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5'-triphosphate (ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 10(6) cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process (p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ (p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced (p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) (p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased (p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe

  16. Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells.

    PubMed Central

    Horie, M; Irisawa, H; Noma, A

    1987-01-01

    1. The adenosine-5'-triphosphate (ATP)-sensitive K+ channel of guinea-pig ventricular cells was examined in the presence and absence of internal Mg2+ or Na+ using an open cell-attached configuration of the patch-clamp technique. 2. Millimolar concentrations of internal Mg2+ ([Mg2+]i) produced marked fluctuations in the outward current, and the amplitude of the open-channel current was reduced with increasing [Mg2+]i. Millimolar Na+ applied internally also decreased the mean amplitude of the outward current, but the increase in current noise was not obvious. These effects became larger when the membrane potential was shifted to be more positive from the K+ equilibrium potential (EK). At potentials negative to EK the inward current was affected by neither internal Mg2+ nor Na+. 3. The external application of Na+, Mg2+ or Ca2+, however, failed to affect the single-channel current. 4. After removal of both internal Mg2+ and Na+, the mean open-channel current-voltage relationship became virtually linear. Referring to these unblocked values, relative amplitudes were determined at different levels of [Mg2+]i or [Na+]i. The dose-response relations gave a Hill coefficient of approximately 1 for Mg2+ block and approximately 2 for Na+ block. The half-maximum concentrations (Kh) for both Mg2+ and Na+ block were shifted to lower values with increasing positive potentials. 5. The power-density spectrum of the open-channel current noise induced by internal Mg2+ showed a Lorentzian function with a corner frequency above 1 kHz, suggesting that the current noise is due to rapid fluctuations of open-channel current between blocked and unblocked states. The corner frequencies gave Mg2+ block and unblock rate constants which were of the order of 10(7) M-1 s-1 and 10(4) s-1, respectively. 6. With increasing external K+ concentration ([K+]o) from 0 to 140 mM the current fluctuations became less prominent, and Kh for Mg2+ block was shifted to higher values. Raising [K+]o enhanced the

  17. 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

  18. Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

    PubMed Central

    Horn, R S; Lystad, E; Adler, A; Walaas, O

    1986-01-01

    When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed. Images Fig. 1. Fig. 3. PMID:3521589

  19. Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2′-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases

    PubMed Central

    Tucker, Kathryn; Lin, Xiaoyan; Kao, C. Cheng; Shaw, Ken; Tan, Hua; Symons, Julian; Behera, Ishani; Rajwanshi, Vivek K.; Dyatkina, Natalia; Wang, Guangyi; Beigelman, Leo

    2015-01-01

    Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities. PMID:26392512

  20. Analysis of Ribonucleotide 5'-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.

    PubMed

    Lu, Gaofei; Bluemling, Gregory R; Collop, Paul; Hager, Michael; Kuiper, Damien; Gurale, Bharat P; Painter, George R; De La Rosa, Abel; Kolykhalov, Alexander A

    2017-03-01

    Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn(2+) is required for enzymatic activity, while Mg(2+) is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

  1. Synthesis of 3'-thioamido-modified 3'-deoxythymidine-5'-triphosphates and their use as chain terminators in Sanger-DNA sequencing.

    PubMed

    Schwarzer, K; Wojczewski, C; Engels, J W

    2001-01-01

    The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxo- propyl)amino]thymidine 1 and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-((6-([(9H-(fluo-ren-9- ylmethoxy)carbonyl]-amino)-1-thioxohexyl)amino) thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5'-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.

  2. Ratiometric detection of adenosine triphosphate (ATP) in water and real-time monitoring of apyrase activity with a tripodal zinc complex.

    PubMed

    Butler, Stephen J

    2014-11-24

    Two tripodal fluorescent probes Zn⋅L(1,2) have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn⋅L(1) allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of several competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.

  3. Diadenosine triphosphate is a novel factor which in combination with cyclodextrins synergistically enhances the biosynthesis of trans-resveratrol in Vitis vinifera cv. Monastrell suspension cultured cells.

    PubMed

    Pietrowska-Borek, Małgorzata; Czekała, Lukasz; Belchí-Navarro, Sarai; Pedreño, María Angeles; Guranowski, Andrzej

    2014-11-01

    Dinucleoside polyphosphates are considered as signal molecules that may evoke response of plant cells to stress. Other compounds whose biological effects have been recognized are cyclodextrins. They are cyclic oligosaccharides that chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack, and they act as true elicitors, since, when added to plant cell culture, they induce the expression of genes involved in some secondary metabolism pathways. Previously, we demonstrated that some dinucleoside polyphosphates triggered the biosynthesis of enzymes involved in the phenylpropanoid pathway in Arabidopsis thaliana. In Vitis vinifera suspension cultured cells, cyclodextrins were shown to enhance the accumulation of trans-resveratrol, one of the basic units of the stilbenes derived from the phenylpropanoid pathway. Here, we show that diadenosine triphosphate, applied alone or in combination with cyclodextrins to the grapevine suspension-cultured cells, increased the transcript level of genes encoding key phenylpropanoid-pathway enzymes as well as the trans-resveratrol production inside cells and its secretion into the extracellular medium. In the latter case, these two compounds acted synergistically. However, the accumulation of trans-resveratrol and its glucoside trans-piceid inside cells were stimulated much better by diadenosine triphosphate than by cyclodextrins.

  4. Purification and properties of the enzymes from Drosophila melanogaster that catalyze the conversion of dihydroneopterin triphosphate to the pyrimidodiazepine precursor of the drosopterins.

    PubMed

    Wiederrecht, G J; Brown, G M

    1984-11-25

    The enzyme system responsible for the conversion of 2-amino-4-oxo-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihyd roptridine triphosphate (dihydroneopterin triphosphate or H2-NTP) to 2-amino-4-oxo-6-acetyl-7,8-dihydro-3H,9H-pyrimido[4,5-b]-[1,4]diazepine (pyrimidodiazepine or PDA), a precursor to the red eye pigments, he drosopterins, has been purified from the heads of Drosophila melanogaster. The PDA-synthesizing system consists of two components, a heat-stable enzyme and a heat-labile enzyme. The heat-stable enzyme can be replaced by sepiapterin synthase A, a previously purified enzyme required for the Mg2+-dependent conversion of H2-NTP to an unstable compound that appears to be 6-pyruvoyltetrahydropterin (pyruvoyl-H4-pterin). The heat-labile enzyme, purified to near-homogeneity and termed PDA synthase (Mr = 48,000), catalyzes the conversion of pyruvoyl-H4-pterin to PDA in a reaction requiring the presence of reduced glutathione. Because PDA is two electrons more reduced than pyruvoyl-H4-pterin, the reducing power required for this transformation is probably supplied by glutathione. The PDA-synthesizing system requires the presence of another thiol-containing compound such as 2-mercaptoethanol when incubation conditions 2-mercaptoethanol is no longer required. Evidence is presented to indicate that the Drosophila eye color mutant, sepia, is missing PDA synthase.

  5. 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.

  6. Discrimination between 8-oxo-2'-deoxyguanosine and 2'-deoxyguanosine in DNA by the single nucleotide primer extension reaction with adap triphosphate.

    PubMed

    Taniguchi, Yosuke; Kikukawa, Yoshiya; Sasaki, Shigeki

    2015-04-20

    The adenosine derivative of 2-oxo-1,3-diazaphenoxazine (Adap) exhibits a superb ability to recognize and form base pairs with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in duplex DNA. In this study, the triphosphate of Adap (dAdapTP) was synthesized and tested for single nucleotide incorporation into primer strands using the Klenow Fragment. The efficiency of dAdapTP incorporation into 8-oxo-dG-containing templates was more than 36-fold higher than with dG-containing templates, and provides better discrimination than does the incorporation of natural 2'-deoxyadenosine triphosphate (dATP). The selective incorporation of dAdapTP into 8-oxo-dG templates was therefore applied to the detection of 8-oxo-dG in human telomeric DNA sequences extracted from H2 O2 -treated HeLa cells. The enzymatic incorporation of dAdapTP into 8-oxo-dG-containing templates may provide a novel basis for sequencing oxidative DNA damage in the genome.

  7. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    SciTech Connect

    Carlow, D.A.; Teh, H.S.; Marth, J.

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  8. A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase.

    PubMed

    Piccirilli, J A; Moroney, S E; Benner, S A

    1991-10-22

    With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

  9. Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.

    PubMed

    Agudo, Rubén; Arias, Armando; Pariente, Nonia; Perales, Celia; Escarmís, Cristina; Jorge, Alberto; Marina, Anabel; Domingo, Esteban

    2008-10-10

    The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.

  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.

  11. Communication: Near edge x-ray absorption fine structure spectroscopy of aqueous adenosine triphosphate at the carbon and nitrogen K-edges.

    PubMed

    Kelly, Daniel N; Schwartz, Craig P; Uejio, Janel S; Duffin, Andrew M; England, Alice H; Saykally, Richard J

    2010-09-14

    Near edge x-ray absorption fine structure (NEXAFS) spectroscopy at the nitrogen and carbon K-edges was used to study the hydration of adenosine triphosphate in liquid microjets. The total electron yield spectra were recorded as a function of concentration, pH, and the presence of sodium, magnesium, and copper ions (Na(+)/Mg(2+)/Cu(2+)). Significant spectral changes were observed upon protonation of the adenine ring, but not under conditions that promote π-stacking, such as high concentration or presence of Mg(2+), indicating that NEXAFS is insensitive to the phenomenon. Intramolecular inner-sphere association of Cu(2+) did create observable broadening of the nitrogen spectrum, whereas outer-sphere association with Mg(2+) did not.

  12. 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.

  13. Visual and light scattering spectrometric method for the detection of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Liang, Lijiao; Zhen, Shujun; Huang, Chengzhi

    2017-02-01

    A highly selective method was presented for colorimetric determination of melamine using uracil 5‧-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

  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. Ultrasensitive method to quantify intracellular zidovudine mono-, di- and triphosphate concentrations in peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry.

    PubMed

    Kinai, Ei; Gatanaga, Hiroyuki; Kikuchi, Yoshimi; Oka, Shinichi; Kato, Shingo

    2015-06-01

    Although zidovudine (AZT) is not the preferred antiretroviral drug for adult HIV-infected patients, it is still widely used in infants for both prevention of mother-to-infant HIV-1 transmission and treatment of HIV-infected children. However, it is difficult to measure intracellular concentrations of AZT metabolites in small blood samples due to their extremely low concentrations in peripheral blood mononuclear cells and interference by endogenous nucleotide triphosphates, residual plasma phosphates and electrolytes. We developed an ultrasensitive assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of intracellular concentrations of zidovudine (AZT)-monophosphate (AZT-MP), -diphosphate (AZT-DP) and -triphosphate (AZT-TP). The high sensitivity was due to the improvement of peripheral blood mononuclear cells extraction for complete removal of plasma and electrolytes, alkalization of LC buffer and use of alkaline-stable high performance liquid chromatography column and tetrabutylammonium hydroxide as the ion pair. Using this method, the lower limits of quantification of AZT, AZT-MP, -DP and -TP were 6, 6, 10 and 10 fmol per sample, respectively. Accuracy ranged 89-115% and precision was lower than 15% in the quantification range of 6-6000 fmol/sample for plasma AZT and intracellular AZT-MP and 10-10 000 fmol/sample for AZT-DP and -TP. The validation parameters met the international requirements. Among nine AZT-treated HIV-infected adult patients, five had low AZT-TP levels (<10 fmol/10(6) cells). Our assay has high sensitivity and is advantageous for evaluation of AZT phosphates in children and infants based on minimum blood sampling requirement.

  16. Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment.

    PubMed

    Guo, Xiaoqing; Dumas, Melanie; Robinson, Bonnie L; Ali, Syed F; Paule, Merle G; Gu, Qiang; Kanungo, Jyotshna

    2017-02-01

    Verapamil is a Ca(2)(+) channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca(2)(+) -permeable N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca(2)(+) entry through L-type Ca(2)(+) channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca(2)(+) channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca(2)(+) release suggesting that ALCAR acts via effectors downstream of Ca(2)(+) . In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca(2)(+) during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  17. 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

    2015-09-07

    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.

  18. Structure–function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins

    PubMed Central

    Gong, Chunling; Smith, Paul; Shuman, Stewart

    2006-01-01

    RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic β-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name “triphosphate tunnel metalloenzyme,” is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5′-end processing. PMID:16809816

  19. Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment

    PubMed Central

    Guo, Xiaoqing; Dumas, Melanie; Robinson, Bonnie L.; Ali, Syed F.; Paule, Merle G.; Gu, Qiang; Kanungo, Jyotshna

    2016-01-01

    Verapamil is a Ca2+ channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca2+-permeable N-methyl-D-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl L-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mM ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mM ALCAR neutralized this effect. ALCAR could reverse ketamine’s effect, possibly through a compensatory mechanism involving extracellular Ca2+ entry through L-type Ca2+ channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca2+ channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca2+ release suggesting that ALCAR acts via effectors downstream of Ca2+. In fact, ALCAR’s protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca2+ during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. PMID:27191126

  20. 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

  1. Flow cytometry and adenosine tri-phosphate analysis: alternative possibilities to evaluate major bacteriological changes in drinking water treatment and distribution systems.

    PubMed

    Vital, Marius; Dignum, Marco; Magic-Knezev, Aleksandra; Ross, Petra; Rietveld, Luuk; Hammes, Frederik

    2012-10-01

    An ever-growing need exists for rapid, quantitative and meaningful methods to quantify and characterize the effect of different treatment steps on the microbiological processes and events that occur during drinking water treatment and distribution. Here we compared cultivation-independent flow cytometry (FCM) and adenosine tri-phosphate (ATP) analysis with conventional cultivation-based microbiological methods, on water samples from two full-scale treatment and distribution systems. The two systems consist of nearly identical treatment trains, but their raw water quality and pre-treatment differed significantly. All of the drinking water treatment processes affected the microbiological content of the water considerably, but once treated, the finished water remained remarkably stable throughout the distribution system. Both the FCM and ATP data were able to describe the microbiology of the systems accurately, providing meaningful process data when combined with other parameters such as dissolved organic carbon analysis. Importantly, the results highlighted a complimentary value of the two independent methods: while similar trends were mostly observed, variations in ATP-per-cell values between water samples were adequately explained by differences in the FCM fingerprints of the samples. This work demonstrates the value of alternative microbial methods for process/system control, optimization and routine monitoring of the general microbial quality of water during treatment and distribution.

  2. Proline modulates the effect of bisphosphonate on calcium levels and adenosine triphosphate production in cell lines derived from bovine Echinococcus granulosus protoscoleces.

    PubMed

    Fuchs, A G; Echeverría, C I; Pérez Rojo, F G; Prieto González, E A; Roldán, E J A

    2014-12-01

    Bisphosphonates have been proposed as pharmacological agents against parasite and cancer cell growth. The effect of these compounds on helminthic cell viability and acellular compartment morphology, however, has not yet been studied. The effects of different types of bisphosphonates, namely etidronate (EHDP), pamidronate (APD), alendronate (ABP), ibandronate (IB) and olpadronate (OPD), and their interaction with amiloride, 1,25-dihydroxycholecalciferol (D3) and proline were evaluated on a cell line derived from bovine Echinococcus granulousus protoscoleces (EGPE) that forms cystic colonies in agarose. The EGPE cell line allowed testing the effect of bisphosphonates alone and in association with other compounds that could modulate calcium apposition/deposition, and were useful in measuring the impact of these compounds on cell growth, cystic colony formation and calcium storage. Decreased cell growth and cystic colony formation were found with EHDP, IB and OPD, and increased calcium storage with EHDP only. Calcium storage in EGPE cells appeared to be sensitive to the effect of amiloride, D3 and proline. Proline decreased calcium storage and increased colony formation. Changes in calcium storage may be associated with degenerative changes of the cysts, as shown in the in vitro colony model and linked to an adenosine triphosphate (ATP) decrease. In conclusion, bisphosphonates could be suitable tempering drugs to treat cestode infections.

  3. 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.

  4. A fluorescent aptasensor for amplified label-free detection of adenosine triphosphate based on core-shell Ag@SiO2 nanoparticles.

    PubMed

    Song, Quanwei; Peng, Manshu; Wang, Le; He, Dacheng; Ouyang, Jin

    2016-03-15

    The novel, facile and universal aptamer-based methods for the highly sensitive and selective fluorescence detection of important biomolecules have attracted considerable interest. Here, we present a label-free aptasensor for adenosine triphosphate (ATP) detection in aqueous solutions by using an ultra-sensitive nucleic acid stain PicoGreen (PG) as a fluorescent indicator and core-shell Ag@SiO2 nanoparticles (NPs) as a metal-enhanced fluorescence (MEF) platform. In the presence of ATP, the complementary DNA (cDNA)/aptamer duplexes confined onto the Ag@SiO2 NPs surface can release their aptamers into the buffered solution, causing a significant reduction in fluorescence intensity. By virtue of the amplified fluorescence signal, this aptasensor toward ATP can achieve a detection limit of 14.2 nM with a wide linear range and exhibit a good assay performance in complex biological samples. This sensing approach is cost-effective and efficient because it avoids the fluorescence labeling process and the use of any enzymes. Hence, this method may offer an alternative tool for determining the concentrations of ATP in biochemical and biomedical research.

  5. 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.

  6. A sensitive quartz crystal microbalance assay of adenosine triphosphate via DNAzyme-activated and aptamer-based target-triggering circular amplification.

    PubMed

    Song, Weiling; Zhu, Zheng; Mao, Yaning; Zhang, Shusheng

    2014-03-15

    In this work, a simple and novel quartz crystal microbalance (QCM) assay is demonstrated to selectively and sensitively detect the adenosine triphosphate (ATP). The amplification process consists of circular nucleic acid strand-displacement polymerization, aptamer recognition strategy and nanoparticle signal amplification. With the involvement of an aptamer-based complex, two amplification reaction templates and AuNP-functionalized probes, the whole circle amplification process is triggered by the target recognition of ATP. As an efficient mass amplifier, AuNP-functionalized probes are introduced to enhance the QCM signals. As a result of DNA multiple amplification, a large number of AuNP-functionalized probes are released and hybridized with the capture probes on the gold electrode. Therefore the QCM signals are significantly enhanced, reaching a detection limit of ATP as low as 1.3 nM. This strategy can be conveniently used for any aptamer-target binding events with other biological detection such as protein and small molecules. Moreover, the practical determination of ATP in cancer cells demonstrates the feasibility of this QCM approach and potential application in clinical diagnostics.

  7. An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate.

    PubMed

    He, Yanlong; Tian, Jianniao; Hu, Kun; Zhang, Juanni; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun; Zhao, Shulin

    2013-11-13

    In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8×10(-12) M to 2.40×10(-4) M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems.

  8. Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

    PubMed

    Martić, Sanela; Rains, Meghan K; Freeman, Daniel; Kraatz, Heinz-Bernhard

    2011-08-17

    The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 μM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

  9. Effects of an ATP analogue, adenosine 5'-[α-thio]-triphosphate, on F1-ATPase rotary catalysis, torque generation, and inhibited intermediated formation.

    PubMed

    Yukawa, Ayako; Watanabe, Rikiya; Noji, Hiroyuki

    2015-03-13

    F1-ATPase (F1), an important rotary motor protein, converts the chemical energy of ATP hydrolysis into mechanical energy using rotary motion with extremely high efficiency. The energy-conversion mechanism for this molecular motor has been extensively clarified by previous studies, which indicate that the interactions between the catalytic residues and the β- and γ-phosphates of ATP are indispensable for efficient catalysis and torque generation. However, the role of α-phosphate is largely unknown. In this study, we observed the rotation of F1 fuelled with an ATP analogue, adenosine 5'-[α-thio]-triphosphate (ATPαS), in which the oxygen has been substituted with a sulfur ion to perturb the α-phosphate/F1 interactions. In doing so, we have revealed that ATPαS does not appear to have any impact on the kinetic properties of the motor or on torque generation compared to ATP. On the other hand, F1 was observed to lapse into the ADP-inhibited intermediate states when in the presence of ATPαS more severely than in the presence of ATP, suggesting that the α-phosphate group of ATP contributes to the avoidance of ADP-inhibited intermediate formation.

  10. Enhanced Production of Adenosine Triphosphate by Pharmacological Activation of Adenosine Monophosphate-Activated Protein Kinase Ameliorates Acetaminophen-Induced Liver Injury.

    PubMed

    Hwang, Jung Hwan; Kim, Yong-Hoon; Noh, Jung-Ran; Choi, Dong-Hee; Kim, Kyoung-Shim; Lee, Chul-Ho

    2015-10-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose.

  11. Evaluation of the Relationship between the Adenosine Triphosphate (ATP) Bioluminescence Assay and the Presence of Bacillus anthracis Spores and Vegetative Cells

    PubMed Central

    Gibbs, Shawn G.; Sayles, Harlan; Colbert, Erica M.; Hewlett, Angela; Chaika, Oleg; Smith, Philip W.

    2014-01-01

    Background: The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. Methods: Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. Results: When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. Conclusions: This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event. PMID:24879485

  12. The use of adenosine and adenosine triphosphate testing in the diagnosis, risk stratification and management of patients with syncope: current evidence and future perspectives.

    PubMed

    Fragakis, Nikolaos; Antoniadis, Antonios P; Saviano, Massimo; Vassilikos, Vassilios; Pappone, Carlo

    2015-03-15

    Syncope is a significant source of cardiovascular-related morbidity yet the etiology is frequently obscure and the identification of patients at highest risk is challenging. Adenosine (AD) and adenosine triphosphate (ATP) administrations have been suggested as potentially useful non-invasive tools in the diagnostic workup of patients with neurally-mediated or bradycardia-related syncope. It has been postulated that both compounds by modulating the autonomic innervation in the heart and exerting negative chronotropic and dromotropic effects in the conduction system, may unmask the mechanism of syncope. However, the clinical implications derived from the efficacy of both tests in the investigation of syncope remain unclear mainly due to inconclusive and occasionally contradictory results of published studies. This review article summarizes recent and past information in the use of ATP and AD in the investigation of syncope with emphasis on clinical trials. We present the current level of evidence for the use of these agents in clinical practice, identify areas where further research is warranted and highlight the future perspectives of these agents as complements to an accurate risk-stratification of patients with syncope.

  13. Live-cell imaging in Caenorhabditis elegans reveals the distinct roles of dynamin self-assembly and guanosine triphosphate hydrolysis in the removal of apoptotic cells.

    PubMed

    He, Bin; Yu, Xiaomeng; Margolis, Moran; Liu, Xianghua; Leng, Xiaohong; Etzion, Yael; Zheng, Fei; Lu, Nan; Quiocho, Florante A; Danino, Dganit; Zhou, Zheng

    2010-02-15

    Dynamins are large GTPases that oligomerize along membranes. Dynamin's membrane fission activity is believed to underlie many of its physiological functions in membrane trafficking. Previously, we reported that DYN-1 (Caenorhabditis elegans dynamin) drove the engulfment and degradation of apoptotic cells through promoting the recruitment and fusion of intracellular vesicles to phagocytic cups and phagosomes, an activity distinct from dynamin's well-known membrane fission activity. Here, we have detected the oligomerization of DYN-1 in living C. elegans embryos and identified DYN-1 mutations that abolish DYN-1's oligomerization or GTPase activities. Specifically, abolishing self-assembly destroys DYN-1's association with the surfaces of extending pseudopods and maturing phagosomes, whereas inactivating guanosine triphosphate (GTP) binding blocks the dissociation of DYN-1 from these membranes. Abolishing the self-assembly or GTPase activities of DYN-1 leads to common as well as differential phagosomal maturation defects. Whereas both types of mutations cause delays in the transient enrichment of the RAB-5 GTPase to phagosomal surfaces, only the self-assembly mutation but not GTP binding mutation causes failure in recruiting the RAB-7 GTPase to phagosomal surfaces. We propose that during cell corpse removal, dynamin's self-assembly and GTP hydrolysis activities establish a precise dynamic control of DYN-1's transient association to its target membranes and that this control mechanism underlies the dynamic recruitment of downstream effectors to target membranes.

  14. Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro.

    PubMed

    Soto, Carolina R; Arroyo, Jorge; Alcayaga, Julio

    2002-01-01

    Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.

  15. 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

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

    2016-06-13

    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 AND 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).

  16. The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5′-Triphosphate towards Influenza A Virus Polymerase

    PubMed Central

    Jin, Zhinan; Smith, Lucas K.; Rajwanshi, Vivek K.; Kim, Baek; Deval, Jerome

    2013-01-01

    T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3′-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus. PMID:23874596

  17. Adsorption and desorption of Cu(II), Cd(II) and Pb(II) ions using chitosan crosslinked with epichlorohydrin-triphosphate as the adsorbent.

    PubMed

    Laus, Rogério; Costa, Thiago G; Szpoganicz, Bruno; Fávere, Valfredo T

    2010-11-15

    In this study, chitosan (CTS) was crosslinked with both epichlorohydrin (ECH) and triphosphate (TPP), by covalent and ionic crosslinking, respectively. The resulting new CTS-ECH-TPP adsorbent was characterized by CHN analysis, EDS, FTIR spectroscopy, TGA and DSC, and the adsorption and desorption of Cu(II), Cd(II) and Pb(II) ions in aqueous solution were investigated. Potentiometric studies were also performed and revealed three titratable protons for each pK(a) value of 5.14, 6.76 and 9.08. The results obtained showed that the optimum pH values for adsorption were 6.0 for Cu(II), 7.0 for Cd(II) and 5.0 for Pb(II). The kinetics study demonstrated that the adsorption process proceeded according to the pseudo-second-order model. Three isotherm models (Langmuir, Freundlich and Dubinin-Radushkevich) were employed in the analysis of the adsorption equilibrium data. The Langmuir model resulted in the best fit and the new adsorbent had maximum adsorption capacities for Cu(II), Cd(II) and Pb(II) ions of 130.72, 83.75 and 166.94 mg g(-1), respectively. Desorption studies revealed that HNO(3) and HCl were the best eluents for desorption of Cu(II), Cd(II) and Pb(II) ions from the crosslinked chitosan.

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

    NASA Astrophysics Data System (ADS)

    Ghadari, Rahim

    2017-01-01

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

  19. Transcranial low-level laser therapy (810 nm) temporarily inhibits peripheral nociception: photoneuromodulation of glutamate receptors, prostatic acid phophatase, and adenosine triphosphate

    PubMed Central

    Pires de Sousa, Marcelo Victor; Ferraresi, Cleber; Kawakubo, Masayoshi; Kaippert, Beatriz; Yoshimura, Elisabeth Mateus; Hamblin, Michael R.

    2016-01-01

    Abstract. Photobiomodulation or low-level light therapy has been shown to attenuate both acute and chronic pain, but the mechanism of action is not well understood. In most cases, the light is applied to the painful area, but in the present study we applied light to the head. We found that transcranial laser therapy (TLT) applied to mouse head with specific parameters (810 nm laser, 300  mW/cm2, 7.2 or 36  J/cm2) decreased the reaction to pain in the foot evoked either by pressure (von Frey filaments), cold, or inflammation (formalin injection) or in the tail (evoked by heat). The pain threshold increasing is maximum around 2 h after TLT, remains up to 6 h, and is finished 24 h after TLT. The mechanisms were investigated by quantification of adenosine triphosphate (ATP), immunofluorescence, and hematoxylin and eosin (H&E) staining of brain tissues. TLT increased ATP and prostatic acid phosphatase (an endogenous analgesic) and reduced the amount of glutamate receptor (mediating a neurotransmitter responsible for conducting nociceptive information). There was no change in the concentration of tubulin, a constituent of the cytoskeleton, and the H&E staining revealed no tissue damage. PMID:26835486

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

    SciTech Connect

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

    2012-06-19

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

  1. Comparison of immunomagnetic separation/adenosine triphosphate rapid method to traditional culture-based method for E. coli and enterococci enumeration in wastewater

    USGS Publications Warehouse

    Bushon, R.N.; Likirdopulos, C.A.; Brady, A.M.G.

    2009-01-01

    Untreated wastewater samples from California, North Carolina, and Ohio were analyzed by the immunomagnetic separation/adenosine triphosphate (IMS/ATP) method and the traditional culture-based method for E. coli and enterococci concentrations. The IMS/ATP method concentrates target bacteria by immunomagnetic separation and then quantifies captured bacteria by measuring bioluminescence induced by release of ATP from the bacterial cells. Results from this method are available within 1 h from the start of sample processing. Significant linear correlations were found between the IMS/ATP results and results from traditional culture-based methods for E. coli and enterococci enumeration for one location in California, two locations in North Carolina, and one location in Ohio (r??values ranged from 0.87 to 0.97). No significant linear relation was found for a second location in California that treats a complex mixture of residential and industrial wastewater. With the exception of one location, IMS/ATP showed promise as a rapid method for the quantification of faecal-indicator organisms in wastewater.

  2. Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System.

    PubMed

    Karalkar, Nilesh B; Leal, Nicole A; Kim, Myong-Sang; Bradley, Kevin M; Benner, Steven A

    2016-07-15

    As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ∼92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ∼86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.

  3. Effects of different concentrations of metal ions on degradation of adenosine triphosphate in common carp (Cyprinus carpio) fillets stored at 4°C: An in vivo study.

    PubMed

    Li, Dapeng; Qin, Na; Zhang, Longteng; Lv, Jian; Li, Qingzheng; Luo, Yongkang

    2016-11-15

    The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx.

  4. Rapid and cost-effective detection of sequence-specific DNA by monitoring the electrochemical response of 2'-deoxyguanosine 5'-triphosphate in a PCR sample.

    PubMed

    Zhang, Xuzhi; Liu, Shufeng; Jiao, Kui; Gao, Hongwei; Shi, Yanjing

    2008-12-01

    This study describes a novel strategy for rapid and cost-effective detection of sequence-specific DNA based upon the essential utility of the polymerase chain reaction (PCR) and electrochemical technologies. A dramatic enhancement of the anodic peak current (i(pa)) and a visible decrease of overpotential towards free 2'-deoxyguanosine 5'-triphosphate (dGTP) could be realized on a glassy carbon electrode modified with short single-walled carbon nanotubes (S-SWNT/GCE). Thereby, the concentration of the free dGTP in the PCR sample mixture could be determined sensitively. The i(pa) of the free dGTP decreased remarkably after a successful PCR amplification owing to the participation of the free dGTP as one of the reactive substrates for the PCR products, namely dsDNA. Based upon this response change of the free dGTP before and after incorporation in PCR, a novel method aiming at detecting PCR results was established. One transgenic maize sample as a model was successfully detected by employing the specific sequences of 35S promoter from cauliflower mosaic virus (CaMV35S) gene and nopaline synthase (NOS) gene as markers. The result was in good accordance with that obtained with gel electrophoresis.

  5. The Therapeutic Potential of Adenosine Triphosphate as an Immune Modulator in the Treatment of HIV/AIDS: A Combination Approach with HAART

    PubMed Central

    Wagner, Marc C.E.

    2011-01-01

    Extracellular adenosine triphosphate (eATP) is a potent molecule that has the capacity to modulate various aspects of cell functions including gene expression. This element of modulation is essential to the role of ATP as a therapeutic agent. The hypothesis presented is that ATP can have an important impact on the treatment of HIV infection. This is supported in part by published research, although a much greater role for ATP is suggested than prior authors ever thought possible. ATP has the ability to enhance the immune system and could thus improve the host’s own defense mechanisms to eradicate the virus-infected cells and restore normal immune function. This could provide effective therapy when used in conjunction with highly active antiretroviral therapies (HAART) to eliminate the latently infected cells. The key lies in applying ATP through the methodology described. This article presents a strategy for using ATP therapeutically along with background evidence to substantiate the importance of using ATP in the treatment of HIV infection. PMID:21675943

  6. Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate

    PubMed Central

    Lee, Mi-Kyung; Kim, Hee-Eun; Park, Eun-Byeol; Lee, Janghyun; Kim, Ki-Hun; Lim, Kyungeun; Yum, Seoyun; Lee, Young-Hoon; Kang, Suk-Jo; Lee, Joon-Hwa; Choi, Byong-Seok

    2016-01-01

    Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation. PMID:27288441

  7. Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces

    PubMed Central

    Ho, Yu-Huai; Wang, Lih-Shinn; Jiang, Hui-Li; Chang, Chih-Hui; Hsieh, Chia-Jung; Chang, Dan-Chi; Tu, Hsin-Yu; Chiu, Tan-Yun; Chao, Huei-Jen; Tseng, Chun-Chieh

    2016-01-01

    Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate “cleanliness” using a sampling area–adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm2, 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm2) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm2, which corresponded to culture-assay levels of <2.5 CFU/cm2. An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate. PMID:27294944

  8. Inositol 1,4,5-triphosphate receptors and NAD(P)H mediate Ca2+ signaling required for hypoxic preconditioning of hippocampal neurons

    PubMed Central

    Bickler, Philip E.; Fahlman, Christian S.; Gray, Jonathan; McKleroy, William; Warren, Daniel E.

    2009-01-01

    Exposure of neurons to a non-lethal hypoxic stress greatly reduces cell death during subsequent severe ischemia (hypoxic preconditioning, HPC). In organotypic cultures of rat hippocampus, we demonstrate that HPC requires inositol triphosphate (IP3) receptor-dependent Ca2+ release from the endoplasmic reticulum (ER) triggered by increased cytosolic NAD(P)H. Ca2+ chelation with intracellular BAPTA, ER Ca2+ store depletion with thapsigargin, IP3 receptor block with xestospongin, and RNA interference against subtype 1 of the IP3 receptor all blunted the moderate increases in [Ca2+]i (50–100 nM) required for tolerance induction. Increases in [Ca2+]i during HPC and neuroprotection following HPC was not prevented with NMDA receptor block or by removing Ca2+ from the bathing medium. Increased NAD(P)H fluorescence in CA1 neurons during hypoxia and demonstration that NADH manipulation increases [Ca2+]i in an IP3R-dependent manner revealed a primary role of cellular redox state in liberation of Ca2+ from the ER. Blockade of IP3Rs and intracellular Ca2+ chelation prevented phosphorylation of known HPC signaling targets, including MAPK p42/44 (ERK), protein kinase B (Akt) and CREB. We conclude that the endoplasmic reticulum, acting via redox/NADH-dependent intracellular Ca2+ store release, is an important mediator of the neuroprotective response to hypoxic stress. PMID:19217932

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

    SciTech Connect

    Leanza, Luigi; Miazzi, Cristina; Ferraro, Paola; Reichard, Peter; Bianchi, Vera

    2010-12-10

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

  10. Comparison of immunomagnetic separation/adenosine triphosphate rapid method to traditional culture-based method for E. coli and enterococci enumeration in wastewater.

    PubMed

    Bushon, Rebecca N; Likirdopulos, Christina A; Brady, Amie M G

    2009-11-01

    Untreated wastewater samples from California, North Carolina, and Ohio were analyzed by the immunomagnetic separation/adenosine triphosphate (IMS/ATP) method and the traditional culture-based method for E. coli and enterococci concentrations. The IMS/ATP method concentrates target bacteria by immunomagnetic separation and then quantifies captured bacteria by measuring bioluminescence induced by release of ATP from the bacterial cells. Results from this method are available within 1h from the start of sample processing. Significant linear correlations were found between the IMS/ATP results and results from traditional culture-based methods for E. coli and enterococci enumeration for one location in California, two locations in North Carolina, and one location in Ohio (r values ranged from 0.87 to 0.97). No significant linear relation was found for a second location in California that treats a complex mixture of residential and industrial wastewater. With the exception of one location, IMS/ATP showed promise as a rapid method for the quantification of faecal-indicator organisms in wastewater.

  11. Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces.

    PubMed

    Ho, Yu-Huai; Wang, Lih-Shinn; Jiang, Hui-Li; Chang, Chih-Hui; Hsieh, Chia-Jung; Chang, Dan-Chi; Tu, Hsin-Yu; Chiu, Tan-Yun; Chao, Huei-Jen; Tseng, Chun-Chieh

    2016-06-09

    Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate "cleanliness" using a sampling area-adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm², 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm²) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm², which corresponded to culture-assay levels of <2.5 CFU/cm². An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate.

  12. Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets†,‡

    PubMed Central

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

    2010-01-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-Å resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer–tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 Å between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

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

    PubMed

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

    2016-10-01

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

  14. Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

    PubMed

    Snowdin, Jacob W; Hsiung, Chia-Heng; Kesterson, Daniel G; Kamath, Vasudeva G; McKee, Edward E

    2015-10-01

    The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

  15. In vivo quantification of active decitabine-triphosphate metabolite: a novel pharmacoanalytical endpoint for optimization of hypomethylating therapy in acute myeloid leukemia.

    PubMed

    Wang, Hongyan; Chen, Ping; Wang, Jiang; Santhanam, Ramasamy; Aimiuwu, Josephine; Saradhi, U V Vijaya; Liu, Zhongfa; Schwind, Sebastian; Mims, Alice; Byrd, John C; Grever, Michael R; Villalona-Calero, Miguel A; Klisovic, Rebecca; Walker, Alison; Garzon, Ramiro; Blum, William; Chan, Kenneth K; Marcucci, Guido

    2013-01-01

    Decitabine (DAC) is used for treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Following cellular uptake, DAC is activated to DAC-triphosphate (TP) and incorporated into DNA. Once incorporated into the DNA, DAC-TP binds and inactivates DNA methyltransferases (DNMTs), thereby leading to hypomethylation and re-expression of epigenetically silenced tumor suppressor genes and ultimately antileukemia activity. However, direct evidence of in vivo DAC-TP occurrence in DAC-treated patients has been difficult to demonstrate due to a lack of suitable validated analytical methodology. Thus, we developed and validated a nonradioactive sensitive and specific LC-MS/MS assay for quantification of DAC-TP. The assay is linear from 50 to 1,000 nM and from 1 to 10 μM and has a lower limit of quantitation of 50 nM and a coefficient of variation for both within- and between-day precision <20%. Following DAC treatment, we detected DAC-TP in parental and DAC-resistant AML cells (in vitro) and bone marrow (BM) and spleen of normal and leukemic mice (in vivo). Downregulation of DNMTs and correlation of DAC-TP concentration with proteins involved in mechanisms of DAC resistance were also demonstrated. The clinical applicability of this method was proven by measuring DAC-TP level in BM and blood mononuclear cells from DAC-treated AML patients. Higher levels are seemingly associated with clinical response. Monitoring the DAC-TP intracellular level may serve as a novel pharmacological endpoint for designing more effective DAC-based regimens.

  16. The CDM superfamily protein MBC directs myoblast fusion through a mechanism that requires phosphatidylinositol 3,4,5-triphosphate binding but is independent of direct interaction with DCrk.

    PubMed

    Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R; Abmayr, Susan M

    2006-12-01

    Myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or "Docker"), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding.

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

    PubMed

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

    2011-09-10

    An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research.

  18. Difference Between Dormant Conduction Sites Revealed by Adenosine Triphosphate Provocation and Unipolar Pace-Capture Sites Along the Ablation Line After Pulmonary Vein Isolation.

    PubMed

    Kogawa, Rikitake; Okumura, Yasuo; Watanabe, Ichiro; Sonoda, Kazumasa; Sasaki, Naoko; Takahashi, Keiko; Iso, Kazuki; Nagashima, Koichi; Ohkubo, Kimie; Nakai, Toshiko; Kunimoto, Satoshi; Hirayama, Atsushi

    2016-01-01

    Dormant pulmonary vein (PV) conduction revealed by adenosine/adenosine triphosphate (ATP) provocation test and exit block to the left atrium by pacing from the PV side of the ablation line ("pace and ablate" method) are used to ensure durable pulmonary vein isolation (PVI). However, the mechanistic relation between ATP-provoked PV reconnection and the unexcitable gap along the ablation line is unclear.Forty-five patients with atrial fibrillation (AF) (paroxysmal: 31 patients, persistent: 14 patients; age: 61.1 ± 9.7 years) underwent extensive encircling PVI (EEPVI, 179 PVs). After completion of EEPVI, an ATP provocation test (30 mg, bolus injection) and unipolar pacing (output, 10 mA; pulse width, 2 ms) were performed along the previous EEPVI ablation line to identify excitable gaps. Dormant conduction was revealed in 29 (34 sites) of 179 PVs (16.2%) after EEP-VI (22/45 patients). Pace capture was revealed in 59 (89 sites) of 179 PVs (33.0%) after EEPVI (39/45 patients), and overlapping sites, ie, sites showing both dormant conduction and pace capture, were observed in 22 of 179 (12.3%) PVs (17/45 patients).Some of the ATP-provoked dormant PV reconnection sites were identical to the sites with excitable gaps revealed by pace capture, but most of the PV sites were differently distributed, suggesting that the main underling mechanism differs between these two forms of reconnection. These findings also suggest that performance of the ATP provocation test followed by the "pace and ablate" method can reduce the occurrence of chronic PV reconnections.

  19. Temperature Dependence of NMR Relaxation Times of Nucleoside Triphosphates and Inorganic Phosphate in the Isolated Perfused Rat Liver. Effect on Pi Compartmentation

    NASA Astrophysics Data System (ADS)

    Dufour, Sylvie; Thiaudière, Eric; Vidal, Giovanni; Gallis, Jean-Louis; Rousse, Nicole; Canioni, Paul

    1996-11-01

    The effect of temperature on31P NMR spectra from isolated perfused rat livers was studied at 9.4 T. Relaxation times (T1andT2) of nucleoside triphosphates (NTP) and inorganic phosphate (Pi) were determined at 37, 25, 15, and 4°C. Under hypothermic conditions, an unexpected apparent line sharpening in the Pi spectral region and a clear emergence of an additional Pi resonance were observed. This additional signal was assigned to mitochondrial Pi.T1values obtained for cytosolic and mitochondrial Pi at 4°C were 1.14 ± 0.24 s (n= 5) and 0.71 ± 0.18 s (n= 5), respectively. No significant mitochondrial contribution to the Pi resonance was observed at 37°C. Quantification of Pi and NTP liver contents at 37 and 4°C was performed by comparing the perfused liver spectrum and the corresponding perchloric acid extract spectrum. Under experimental conditions of low external Pi (0.12 mM), it was concluded that intracellular Pi was completely NMR-visible at 4 and 37°C. The observation of the mitochondrial Pi signal at 4°C was well explained by an increase in the Pi level within the matrix, in response to the mitochondrial swelling induced by hypothermia, as observed by electron microscopy.T2values for the cytosolic Pi at 37 and 4°C were 17 ± 4 ms (n= 8) and 22 ± 4 ms (n= 10), respectively. Comparison with measured linewidths indicated that line broadening for the main phosphorylated metabolites-including matrix Pi-was the result ofB0field inhomogeneity. The additional broadening of the cytosolic Pi resonance at 4 and 37°C was attributed to pH heterogeneity within the liver.

  20. Mechanosensitive release of adenosine 5'-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain.

    PubMed

    Beckel, Jonathan M; Argall, Arthur J; Lim, Jason C; Xia, Jingsheng; Lu, Wennan; Coffey, Erin E; Macarak, Edward J; Shahidullah, Mohammed; Delamere, Nicholas A; Zode, Gulab S; Sheffield, Val C; Shestopalov, Valery I; Laties, Alan M; Mitchell, Claire H

    2014-09-01

    As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

  1. Supplementation of Exogenous Adenosine 5′-Triphosphate Enhances Mechanical Properties of 3D Cell–Agarose Constructs for Cartilage Tissue Engineering

    PubMed Central

    Gadjanski, Ivana; Yodmuang, Supansa; Spiller, Kara; Bhumiratana, Sarindr

    2013-01-01

    Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5′-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure–function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct. PMID:23651296

  2. Multidrug Resistance-Associated Protein 2 Expression Is Upregulated by Adenosine 5’-Triphosphate in Colorectal Cancer Cells and Enhances Their Survival to Chemotherapeutic Drugs

    PubMed Central

    Vinette, Valérie; Placet, Morgane; Arguin, Guillaume; Gendron, Fernand-Pierre

    2015-01-01

    Extracellular adenosine 5’-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity. PMID:26295158

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

    PubMed Central

    Cai, Yiying; Leck, Hui; Lim, Tze Peng; Teo, Jocelyn; Lee, Winnie; Hsu, Li Yang; Koh, Tse Hsien; Tan, Thuan Tong; Tan, Thean-Yen; Kwa, Andrea Lay-Hoon

    2015-01-01

    Background Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations. Methods 100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (105 CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (TRLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of TRLU values was determined. External validation was further done using 50 additional CR-GNB. Results Predictive accuracies of TRLU were high for when all antibiotic combinations and species were collectively analyzed (TRLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (TRLU = 0.81, UA = 91%), and lowest for AB (TRLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively. Conclusion We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations. PMID:26460891

  4. Functional defect of variants in the adenosine triphosphate-binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX-770).

    PubMed

    Delaunay, Jean-Louis; Bruneau, Alix; Hoffmann, Brice; Durand-Schneider, Anne-Marie; Barbu, Véronique; Jacquemin, Emmanuel; Maurice, Michèle; Housset, Chantal; Callebaut, Isabelle; Aït-Slimane, Tounsia

    2017-02-01

    ABCB4 (MDR3) is an adenosine triphosphate (ATP)-binding cassette (ABC) transporter expressed at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene are responsible for several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ABCB4 missense variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Five disease-causing variations in these motifs have been identified in ABCB4 (G535D, G536R, S1076C, S1176L, and G1178S), three of which are homologous to the gating mutations of cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7; i.e., G551D, S1251N, and G1349D), that were previously shown to be function defective and corrected by ivacaftor (VX-770; Kalydeco), a clinically approved CFTR potentiator. Three-dimensional structural modeling predicted that all five ABCB4 variants would disrupt critical interactions in the binding of ATP and thereby impair ATP-induced nucleotide-binding domain dimerization and ABCB4 function. This prediction was confirmed by expression in cell models, which showed that the ABCB4 mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. As also hypothesized on the basis of molecular modeling, PC secretion activity of the mutants was rescued by the CFTR potentiator, ivacaftor (VX-770).

  5. Do Cyclosporine A, an IL-1 Receptor Antagonist, Uridine Triphosphate, Rebamipide, and/or Bimatoprost Regulate Human Meibomian Gland Epithelial Cells?

    PubMed Central

    Kam, Wendy R.; Liu, Yang; Ding, Juan; Sullivan, David A.

    2016-01-01

    Purpose Researchers have hypothesized that treatment with cyclosporine A (CyA), interleukin-1 receptor antagonists (IL-1RA; e.g., anakinra), P2Y2 receptor agonists (e.g., uridine triphosphate; UTP), and rebamipide may alleviate human meibomian gland dysfunction (MGD) and/or dry eye disease. Investigators have also proposed that prostaglandin analogues (e.g., bimatoprost) may induce MGD. Our goal was to determine whether these compounds directly influence human meibomian gland epithelial cell (HMGEC) function. Methods Multiple concentrations of each compound were tested for effects on immortalized (I) HMGEC morphology and survival. Nontoxic dosages were used for our studies. Immortalized HMGEC were cultured in the presence of vehicle, CyA, IL-1RA, UTP, rebamipide, or bimatoprost for up to 6 days in various media. Experiments included positive controls for proliferation (epidermal growth factor and bovine pituitary extract), differentiation (azithromycin), and signaling pathway activation (insulin-like growth factor 1). Cells were analyzed for neutral lipid staining, lysosome accumulation, lipid composition, and phosphatidylinositol-3-kinase/Akt (AKT), phosphorylation. Results Our findings demonstrate that CyA, IL-1RA, UTP, rebamipide, and bimatoprost had no effect on the proliferation; neutral lipid content; lysosome number; or levels of free cholesterol, triglycerides, or phospholipids in IHMGECs. Cylosporine A, IL-1RA, rebamipide, and bimatoprost significantly reduced the phosphorylation of AKT, as compared to control. Of interest, tested doses of CyA above 8 nM killed the IHMGECs. Conclusions Our results show that CyA, IL-1RA, UTP, rebamipide, and bimatoprost do not influence the proliferation or differentiation of IHMGEC. However, with the exception of UTP, these compounds do decrease the activity of the AKT signaling pathway, which is known to promote cell survival. PMID:27552406

  6. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    PubMed

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.

  7. Effects of chronic digitalization on cardiac and renal Na+ + K+-dependent adenosine triphosphate activity and circulating catecholamines in the dog.

    PubMed

    Nechay, B R; Jackson, R E; Ziegler, M G; Neldon, S L; Thompson, J D

    1981-09-01

    To extend our understanding of the mechanism of action of digitalis drugs, we studied electrocardiograms (ECGs), renal function, plasma concentrations of catecholamines, and myocardial and renal Na+ + K+-dependent adenosine triphosphate (Na+ + K+ ATPase) activity in chronically digitalized dogs. Five healthy, male, mongrel dogs received a therapeutic regimen of digoxin (0.1 mg/kg on day 1 in three divided doses followed by 0.025 mg/kg per day) orally for 2-4 months. This resulted in plasma digoxin concentrations of 1.1 to 4.7 ng/ml as determined by radioimmunoassay. Six control dogs received daily gelatin capsules by mouth. ECGs monitored throughout the study showed no changes. Digitalized dogs had elevated plasma norepinephrine concentrations (347 vs. 137 pg/ml in controls) and no change in plasma epinephrine concentrations. Digitalized dogs had elevated glomerular filtration rates (0.74 vs. 0.94 ml/min per g of kidney) without significant changes in renal handling of electrolytes and water. All of the above studies were done without the aid of restraining drugs or infusions. The animals were killed with an overdose of pentobarbital for in vitro studies. In digitalized dogs, microsomal Na+ + K+ ATPase-specific activity was 26 to 33% lower in the renal cortex, medulla, and papilla, and 46% lower in the cardiac left ventricle than in control dogs. Digitalization did not alter the osmolalities of renal tissues. We conclude that chronic reduction Na+ + K+ ATPase activity by one-third dose does not cause abnormalities in renal handling of electrolytes and water, and inhibition of Na+ + K+ ATPase in the left ventricular muscle by one-half is associated with no obvious ECG changes in the dog. Further, elevated plasma norepinephrine concentrations may contribute to both the therapeutic and the toxic effects of digitalis.

  8. Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5',5'-triphosphate bridge.

    PubMed

    Zytek, Malgorzata; Kowalska, Joanna; Lukaszewicz, Maciej; Wojtczak, Blazej A; Zuberek, Joanna; Ferenc-Mrozek, Aleksandra; Darzynkiewicz, Edward; Niedzwiecka, Anna; Jemielity, Jacek

    2014-12-07

    A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,β-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m3(2,2,7)GppNHpG) or β-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.

  9. Functional and structural characterization of DR_0079 from Deinococcus radiodurans, a novel Nudix hydrolase with a preference for cytosine (deoxy) ribonucleoside 5'-di- and triphosphates

    SciTech Connect

    Buchko, Garry W.; Litvinova, Olga; Robinson, Howard; Yakunin, Alexander F.; Kennedy, Michael A.

    2008-06-24

    The Deinococcus radiodurans Nudix hydrolase DR0079 was assayed for activity towards a wide variety of substrates and observed to have a marked specificity for cytosine ribonucleoside 5’-diphosphate (CDP) and cytosine ribonucleoside 5’-triphosphate (CTP) with a slight preference for CDP. The next most specific substrates, with a relative activity of <50%, were the corresponding deoxyribose nucleosides, dCDP and dCTP. Enzyme hydrolase activity at the site of the phosphodiester bond was corroborated using 31P NMR spectroscopy to follow the phosphorus resonances for two substrates, CDP and IDP, and the hydrolysis products, NMP and Pi. Optimum activity for CDP was determined to be at pH 9.0 – 9.5. The optimal divalent cation for CDP hydrolysis at this pH was Mg2+ followed by Mn2+ (~47%) and Co2+(~27%). The biochemical data is discussed with reference to the crystal structure for the D. radiodurans DR0079 that was determined in the apo-metal form at 1.9 Å resolution. The protein in the crystal structure contains nine β-strands, three α-helices, and two 3-10 helices that are organized into three subdomains; an N-terminal β-sheet, a central Nudix core, and a C-terminal helixturn- helix motif. As observed for all known structures of Nudix hydrolases, the α-helix of the ‘Nudix box’ is one of two helices that sandwich a ‘four-strand’ mixed β-sheet. Using 15N-labelled DR0079, NMR chemical shift mapping experiments were performed with the paramagnetic divalent cation Co2+ and the non-hydrolyzable substrate thymidine- 5’-O-(α,β-methylenediphosphate (TMP-CP). The results of the chemical shift perturbation experiments were mapped onto the crystal structure of DR0079 and a model for substrate binding proposed.

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

    SciTech Connect

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

    1987-03-05

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

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

    PubMed

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

    1987-03-05

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H2O by D2O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex.

  12. In situ amplified electrochemical aptasensing for sensitive detection of adenosine triphosphate by coupling target-induced hybridization chain reaction with the assembly of silver nanotags.

    PubMed

    Zhou, Qian; Lin, Youxiu; Lin, Yuping; Wei, Qiaohua; Chen, Guonan; Tang, Dianping

    2016-01-01

    Biomolecular immobilization and construction of the sensing platform are usually crucial for the successful development of a high-efficiency detection system. Herein we report on a novel and label-free signal-amplified aptasensing for sensitive electrochemical detection of small molecules (adenosine triphosphate, ATP, used in this case) by coupling with target-induced hybridization chain reaction (HCR) and the assembly of electroactive silver nanotags. The system mainly consisted of two alternating hairpin probes, a partial-pairing trigger-aptamer duplex DNA and a capture probe immobilized on the electrode. Upon target ATP introduction, the analyte attacked the aptamer and released the trigger DNA, which was captured by capture DNA immobilized on the electrode to form a newly partial-pairing double-stranded DNA. Thereafter, the exposed domain at trigger DNA could be utilized as the initator strand to open the hairpin probes in sequence, and propagated a chain reaction of hybridization events between two alternating hairpins to form a long nicked double-helix. The electrochemical signal derived from the assembled silver nanotags on the nicked double-helix. Under optimal conditions, the electrochemical aptasensor could exhibit a high sensitivity and a low detection limit, and allowed the detection of ATP at a concentration as low as 0.03 pM. Our design showed a high selectivity for target ATP against its analogs because of the high-specificity ATP-aptamer reaction, and its applicable for monitoring ATP in the spiking serum samples. Improtantly, the distinct advantages of the developed aptasensor make it hold a great potential for the development of simple and robust sensing strategies for the detection of other small molecules by controlling the apatmer sequence.

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

    PubMed Central

    2013-01-01

    Background Currently, there is a lack of studies examining the effects of adenosine-5′-triphosphate (ATP) supplementation utilizing a long-term, periodized resistance-training program (RT) in resistance-trained populations. Therefore, we investigated the effects of 12 weeks of 400 mg per day of oral ATP on muscular adaptations in trained individuals. We also sought to determine the effects of ATP on muscle protein breakdown, cortisol, and performance during an overreaching cycle. Methods The study was a 3-phase randomized, double-blind, and placebo- and diet-controlled intervention. Phase 1 was a periodized resistance-training program. Phase 2 consisted of a two week overreaching cycle in which volume and frequency were increased followed by a 2-week taper (Phase 3). Muscle mass, strength, and power were examined at weeks 0, 4, 8, and 12 to assess the chronic effects of ATP; assessment performance variables also occurred at the end of weeks 9 and 10, corresponding to the mid and endpoints of the overreaching cycle. Results There were time (p < 0.001), and group x time effects for increased total body strength (+55.3 ± 6.0 kg ATP vs. + 22.4 ± 7.1 kg placebo, p < 0.001); increased vertical jump power (+ 796 ± 75 ATP vs. 614 ± 52 watts placebo, p < 0.001); and greater ultrasound determined muscle thickness (+4.9 ± 1.0 ATP vs. (2.5 ± 0.6 mm placebo, p < 0.02) with ATP supplementation. During the overreaching cycle, there were group x time effects for strength and power, which decreased to a greater extent in the placebo group. Protein breakdown was also lower in the ATP group. Conclusions Our results suggest oral ATP supplementation may enhance muscular adaptations following 12-weeks of resistance training, and prevent decrements in performance following overreaching. No statistically or clinically significant changes in blood chemistry or hematology were observed. Trial registration ClinicalTrials.gov NCT01508338 PMID

  14. Effects of wortmannin, sodium nitroprusside, insulin, genistein, and guanosine triphosphate on chemotaxis and cell growth of Entodinium caudatum, Epidinium caudatum, and mixed ruminal protozoa.

    PubMed

    Diaz, H L; Knapp, J R; Karnati, S K R; Dehority, B A; Firkins, J L

    2014-01-01

    The mechanisms by which ruminal protozoa sense and migrate toward nutrients are not fully understood. Chemotaxis by many diverse eukaryotic cells is mediated by phosphatidylinositol-3-kinase, which is highly conserved in receptor tyrosine kinase (RTK) signaling pathways and consistently inhibited by wortmannin. In experiment 1a, increasing the concentration of wortmannin inhibited cell growth nonlinearly at 24h of a culture of the rumen protozoan Entodinium caudatum, but high variability prevented growth inhibition of Epidinium caudatum from reaching significance. In experiment 1b, increasing the insulin concentration recovered 24-h cell counts for both cultures, depending on wortmannin concentration. In experiment 2, addition of sodium nitroprusside (Snp; activator of protein kinase G for cilial beat reversal in nonrumen ciliate models) at 500µM or wortmannin at 200µM in beakers containing rumen fluid decreased random swimming by mixed entodiniomorphids into capillary tubes (inserted into beakers) containing saline. Both Snp and wortmannin increased chemotaxis into tubes containing glucose compared with the beaker control. For isotrichids, beaker treatments had no response. Glucose increased chemotaxis, but peptides decreased chemotaxis even when combined with glucose. In experiment 3, we assessed preincubation of genistein (a purported RTK blocker in nonrumen ciliate models) at 40 or 400µM in beakers and guanosine triphosphate (GTP; a universal chemorepellent in nonrumen ciliate models, perhaps mediated through an RTK) at 10 or 100µM combined with glucose in capillary tubes. Neither genistein nor GTP affected chemotaxis toward glucose for entodiniomorphids. However, GTP at 100µM reduced chemotaxis toward glucose for isotrichids. After the animal is fed, isotrichids that are depleted in glycogen migrate to the dorsal area of the rumen, and the rapid uptake of sugars is enhanced through strong chemotaxis but can be reversed by peptides or GTP. In contrast

  15. Effect of extracellular adenosine 5'-triphosphate on cryopreserved epididymal cat sperm intracellular ATP concentration, sperm quality, and in vitro fertilizing ability.

    PubMed

    Thuwanut, Paweena; Arya, Nlin; Comizzoli, Pierre; Chatdarong, Kaywalee

    2015-09-15

    Intracellular adenosine 5'-triphosphate (ATP) is essential for supporting sperm function in the fertilization process. During cryopreservation, damage of sperm mitochondrial membrane usually leads to compromised production of intracellular ATP. Recently, extracellular ATP (ATPe) was introduced as a potent activator of sperm motility and fertilizing ability. This study aimed to evaluate (1) levels of intracellular ATP in frozen-thawed epididymal cat sperm after incubation with ATPe and (2) effects of ATPe on epididymal cat sperm parameters after freezing and thawing. Eighteen male cats were included. For each replicate, epididymal sperm from two cats were pooled to one sample (N = 9). Each pooled sample was cryopreserved with the Tris-egg yolk extender into three straws. After thawing, the first and second straws were incubated with 0-, 1.0-, or 2.5-mM ATPe for 10 minutes and evaluated for sperm quality at 10 minutes, 1, 3, and 6 hours after thawing and fertilizing ability. The third straw was evaluated for intracellular ATP concentration in control and with 2.5-mM ATPe treatment. Higher concentration of intracellular sperm ATP was observed in the samples treated with 2.5-mM ATPe compared to the controls (0.339 ± 0.06 μg/2 × 10(6) sperm vs. 0.002 ± 0.003 μg/2 × 10(6) sperm, P ≤ 0.05). In addition, incubation with 2.5-mM ATPe for 10 minutes promoted sperm motility (56.7 ± 5.0 vs. 53.3 ± 4.4%, P ≤ 0.05) and progressive motility (3.1 ± 0.2 vs. 2.8 ± 0.4, P ≤ 0.05), mitochondrial membrane potential (36.4 ± 5.5 vs. 28.7 ± 4.8%, P ≤ 0.05), and blastocyst rate (36.1 ± 7.0 and 28.8 ± 7.4%, P ≤ 0.05) compared with the controls. In contrast, ATPe remarkably interfered acrosome integrity after 6 hours of postthawed incubation. In sum, the present finding that optimal incubation time of postthaw epididymal cat sperm under proper ATPe condition might constitute a rationale for the studies on other endangered wild felids regarding sperm quality and embryo

  16. Intracellular cytarabine triphosphate production correlates to deoxycytidine kinase/cytosolic 5'-nucleotidase II expression ratio in primary acute myeloid leukemia cells.

    PubMed

    Yamauchi, Takahiro; Negoro, Eiju; Kishi, Shinji; Takagi, Kazutaka; Yoshida, Akira; Urasaki, Yoshimasa; Iwasaki, Hiromichi; Ueda, Takanori

    2009-06-15

    Cytarabine (ara-C) is the key agent for treating acute myeloid leukemia (AML). After being transported into leukemic cells by human equilibrative nucleoside transporter 1 (hENT1), ara-C is phosphorylated to ara-C triphosphate (ara-CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase II (cN-II) are associated with the production of ara-CTP. Because ara-C's cytotoxicity depends on ara-CTP production, parameters that are most related to ara-CTP formation would predict ara-C sensitivity and the clinical outcome of ara-C therapy. The present study focused on finding any correlation between the capacity to produce ara-CTP and ara-C-metabolizing factors. In vitro ara-CTP production, mRNA levels of hENT1, dCK, and cN-II, and ara-C sensitivity were evaluated in 34 blast samples from 33 leukemic patients including 26 with AML. A large degree of heterogeneity was seen in the capacity to produce ara-CTP and in mRNA levels of hENT1, dCK, and cN-II. Despite the lack of any association between each of the transcript levels and ara-CTP production, the ratio of dCK/cN-II transcript levels correlated significantly with the amount of ara-CTP among AML samples. The HL-60 cultured leukemia cell line and its three ara-C-resistant variants (HL-60/R1, HL-60/R2, HL-60/R3), which were 8-, 10-, and 500-fold more resistant than HL-60, respectively, were evaluated similarly. The dCK/cN-II ratio was again proportional to ara-CTP production and to ara-C sensitivity. The dCK/cN-II ratio may thus predict the capacity for ara-CTP production and ultimately, ara-C sensitivity in AML.

  17. Adenosine triphosphate stress 99mTc-methoxyisobutylisonitrile gated myocardial perfusion imaging efficacy in diagnosing stent restenosis following coronary stent implantation

    PubMed Central

    Zhang, Pengfei; Chen, Song; Li, Yang; Du, Qiuhong; Wang, Lijuan; Sun, Yingxian; Li, Yaming

    2016-01-01

    Coronary stent restenosis rate following implantation is considerably high. The adenosine stress gated myocardial perfusion imaging (G-MPI) method has been widely used in the diagnosis, risk stratification and prognosis evaluation of coronary heart disease; however, the high cost of adenosine limits its clinical application. The aim of the present study was to investigate the efficacy of adenosine triphosphate (ATP) stress 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) G-MPI for diagnosis in-stent restenosis following coronary stent implantation. Data from 66 patients with typical angina pectoris symptoms who had undergone percutaneous coronary stent implantation >3 months prior to participation in the study were analyzed. All the patients underwent ATP stress 99mTc-MIBI G-MPI and coronary artery angiography as the criterion diagnostic standard within 1 month. The sensitivity, specificity, and accuracy of ATP stress 99mTc-MIBI G-MPI in the assessment of in-stent restenosis were calculated. In addition, Fisher's exact probability methods were used to compare differences between experimental groups. Among 66 patients with a total of 99 implanted coronary arterial branches, 39 patients (59%) with 45 coronary arteries (45%) presented in-stent restenosis. The diagnostic sensitivity, specificity, accuracy, positive predictive and negative predictive value of ATP stress 99mTc-MIBI G-MPI for assessing stent restenosis in all patients were 85, 89, 86, 92 and 80%, respectively. Similarly, these values in patients with myocardial infarction were 79, 88, 83, 88 and 78%, respectively, while in patients without myocardial infarction the values were 90, 91, 90, 95 and 83%, respectively. Therefore, the diagnostic efficacy of ATP stress 99mTc-MIBI G-MPI in patients without myocardial infarction was higher compared with those with myocardial infarction; however, no significant difference was observed between the two groups. Furthermore, the sensitivity, specificity and accuracy for

  18. Adenosine triphosphate stress (99m)Tc-methoxyisobutylisonitrile gated myocardial perfusion imaging efficacy in diagnosing stent restenosis following coronary stent implantation.

    PubMed

    Zhang, Pengfei; Chen, Song; Li, Yang; Du, Qiuhong; Wang, Lijuan; Sun, Yingxian; Li, Yaming

    2016-12-01

    Coronary stent restenosis rate following implantation is considerably high. The adenosine stress gated myocardial perfusion imaging (G-MPI) method has been widely used in the diagnosis, risk stratification and prognosis evaluation of coronary heart disease; however, the high cost of adenosine limits its clinical application. The aim of the present study was to investigate the efficacy of adenosine triphosphate (ATP) stress (99m)Tc-methoxyisobutylisonitrile ((99m)Tc-MIBI) G-MPI for diagnosis in-stent restenosis following coronary stent implantation. Data from 66 patients with typical angina pectoris symptoms who had undergone percutaneous coronary stent implantation >3 months prior to participation in the study were analyzed. All the patients underwent ATP stress (99m)Tc-MIBI G-MPI and coronary artery angiography as the criterion diagnostic standard within 1 month. The sensitivity, specificity, and accuracy of ATP stress (99m)Tc-MIBI G-MPI in the assessment of in-stent restenosis were calculated. In addition, Fisher's exact probability methods were used to compare differences between experimental groups. Among 66 patients with a total of 99 implanted coronary arterial branches, 39 patients (59%) with 45 coronary arteries (45%) presented in-stent restenosis. The diagnostic sensitivity, specificity, accuracy, positive predictive and negative predictive value of ATP stress (99m)Tc-MIBI G-MPI for assessing stent restenosis in all patients were 85, 89, 86, 92 and 80%, respectively. Similarly, these values in patients with myocardial infarction were 79, 88, 83, 88 and 78%, respectively, while in patients without myocardial infarction the values were 90, 91, 90, 95 and 83%, respectively. Therefore, the diagnostic efficacy of ATP stress (99m)Tc-MIBI G-MPI in patients without myocardial infarction was higher compared with those with myocardial infarction; however, no significant difference was observed between the two groups. Furthermore, the sensitivity, specificity and

  19. Pharmacokinetics of lamivudine and lamivudine-triphosphate after administration of 300 milligrams and 150 milligrams once daily to healthy volunteers: results of the ENCORE 2 study.

    PubMed

    Else, Laura J; Jackson, Akil; Puls, Rebekah; Hill, Andrew; Fahey, Paul; Lin, Enmoore; Amara, Alieu; Siccardi, Marco; Watson, Victoria; Tjia, John; Emery, Sean; Khoo, Saye; Back, David J; Boffito, Marta

    2012-03-01

    There is interest in evaluating the efficacy of lower doses of certain antiretrovirals for clinical care. We determined here the bioequivalence of plasma lamivudine (3TC) and intracellular 3TC-triphosphate (3TC-TP) concentrations after the administration of two different doses. ENCORE 2 was a randomized crossover study. Subjects received 3TC at 300 and 150 mg once daily for 10 days (arm 1; n = 13) or vice versa (arm 2; n = 11), separated by a 10-day washout. Pharmacokinetic (PK) profiles (0 to 24 h) were assessed on days 10 and 30. Plasma 3TC and 3TC-TP levels in peripheral blood mononuclear cells were quantified by high-performance liquid chromatography-tandem mass spectrometry. Within-subject changes in PK parameters (the area under the concentration-time curve from 0 to 24 h [AUC(0-24)], the trough concentration of drug in plasma at 24 h [C(24)], and the maximum concentration of drug in plasma [C(max)]) were evaluated by determining the geometric mean ratios (GMRs) adjusted for study arm, period, and intra-individual variation. Regimens were considered bioequivalent if the 90% confidence interval (90% CI) fell within the range of 0.8 to 1.25. A total of 24 subjects completed the study. The GM (90% CI) 3TC AUC(0-24)), expressed as ng·h/ml, for the 300- and 150-mg doses were 8,354 (7,609 to 9,172) and 4,773 (4,408 to 5,169), respectively. Bioequivalence in 3TC PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.57 (0.55 to 0.60), 0.63 (0.59 to 0.67), and 0.56 (0.53 to 0.60), respectively. The GM (90% CI) 3TC-TP AUC(0-24) values (pmol·h/10(6) cells) for the 300- and 150-mg doses were 59.5 (51.8 to 68.3) and 44.0 (38.0 to 51.0), respectively. Bioequivalence in 3TC-TP PK following the administration of 300 and 150 mg was not demonstrated: the GMRs for AUC(0-24), C(24), and C(max) were 0.73 (0.64 to 0.83), 0.82 (0.68 to 0.99), and 0.70 (0.61 to 0.82), respectively. We found that 3TC at 150 mg is

  20. Activation or block of adenosine triphosphate-sensitive potassium channel have opposite effects on postcardioplegic myocardial dysfunction, "stunning". A multivariate prediction based on relative operating characteristic curve.

    PubMed

    Puddu, P E; Sugimoto, S; Monti, F; Iwashiro, K; Dawodu, A A; Schiariti, M; Chiavarelli, R; Marino, B; Campa, P P

    1995-09-01

    The relative effects of nicotinic acid (NA) and nitroglycerin (NT) added to cold high K+ cardioplegia were studied, to represent the two moieties of the adenosine triphosphate-sensitive potassium channel (KATP) activator nicorandil (N). In addition, we made a pooled analysis of a large series of experiments performed in our Laboratory to investigate the effects of KATP activation by N, or block (by glibenclamide, G), on postcardioplegic myocardial dysfunction. In both studies, reversibility from myocardial dysfunction (stunning) was assessed by the positive inotropic agent dobutamine. Guinea pig papillary muscle preparations were immersed in Tyrode's solution (O2 content 16 ml/l, 37 degrees C), then hypoxic (O2 content 5 ml/l) superfusion with hypothermic (20 degrees C) cardioplegic Saint Thomas' Hospital solution (STHS) was performed for 120 min. We investigated: A) 5 groups based on treatments added to STHS: 1) saline (Control (C)); 2) N = 1 mmol/L; 3) G = 1 mumol/L (also given for 15 min in Tyrode's solution); 4) NA = 1 mmol/L; 5) NT = 100 mumol/L; B) 76 consecutive experiments and we defined, independent of whether just before or during STHS: 1) KATP activation (by N, in the concentration range 1 mumol/L to 1 mmol/L, n = 36); 2) KATP block (by G 1 mumol/L, either alone or just before N, n = 20); 3) controls (n = 20) (either saline, n = 12, or saline plus dimethyl sulfoxide, as vehicle, at the ratio 100 to 1, n = 8). Absolute isometric contractility variables were evaluated along with percent changes of baseline values: 1) at 30 s of STHS, 2) after 60 min of reoxygenation with Tyrode's solution and 3) following further 15 min of dobutamine 10 mumol/L. In all preparations, developed tension (DT), time to peak tension (TPT), DT/TPT and time to arrest (TTA) were measured. In study A): TTA was significantly abbreviated (intergroup F = 5.79, p < 0.001) in N (49 +/- 11 s, mean +/- SD) p < 0.01 vs C and NA). At 30 s of STHS %DT/TPT was unchanged among groups. By

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

    USGS Publications Warehouse

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

    2009-01-01

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

  2. Microbial Group Specific Uptake Kinetics of Inorganic Phosphate and Adenosine-5′-Triphosphate (ATP) in the North Pacific Subtropical Gyre

    PubMed Central

    Björkman, Karin; Duhamel, Solange; Karl, David M.

    2012-01-01

    We investigated the concentration dependent uptake of inorganic phosphate (Pi) and adenosine-5′-triphosphate (ATP) in microbial populations in the North Pacific Subtropical Gyre (NPSG). We used radiotracers to measure substrate uptake into whole water communities, differentiated microbial size classes, and two flow sorted groups; Prochlorococcus (PRO) and non-pigmented bacteria (NPB). The Pi concentrations, uptake rates, and Pi pool turnover times (Tt) were (mean, ±SD); 54.9 ± 35.0 nmol L−1 (n = 22), 4.8 ± 1.9 nmol L−1 day−1 (n = 19), and 14.7 ± 10.2 days (n = 19), respectively. Pi uptake into >2 μm cells was on average 12 ± 7% (n = 15) of the total uptake. The kinetic response to Pi (10–500 nmol L−1) was small, indicating that the microorganisms were close to their maximum uptake velocity (Vmax). Vmax averaged 8.0 ± 3.6 nmol L−1 day−1 (n = 19) in the >0.2 μm group, with half saturation constants (Km) of 40 ± 28 nmol L−1 (n = 19). PRO had three times the cell specific Pi uptake rate of NPB, at ambient concentrations, but when adjusted to cells L−1 the rates were similar, and these two groups were equally competitive for Pi. The Tt of γ-P-ATP in the >0.2 μm group were shorter than for the Pi pool (4.4 ± 1.0 days; n = 6), but this difference diminished in the larger size classes. The kinetic response to ATP was large in the >0.2 μm class with Vmax exceeding the rates at ambient concentrations (mean 62 ± 27 times; n = 6) with a mean Vmax for γ-P-ATP of 2.8 ± 1.0 nmol L−1 day−1, and Km at 11.5 ± 5.4 nmol L−1 (n = 6). The NPB contribution to γ-P-ATP uptake was high (95 ± 3%, n = 4) at ambient concentrations but decreased to ∼50% at the highest ATP amendment. PRO had Km values 5–10 times greater than NPB. The above indicates that PRO and NPB were in close competition in terms of Pi acquisition

  3. Spatial and Temporal Variability in the Concentration and Turnover of the Inorganic Phosphate and Adenosine-5'-triphosphate pools in the North Pacific Subtropical Gyre

    NASA Astrophysics Data System (ADS)

    Björkman, Karin; Church, Matthew; Karl, David

    2015-04-01

    The microbial community's utilization of inorganic phosphate (Pi) and adenosine-5'-triphosphate (ATP) as a function of the Pi pool concentration was studied over a multi-year period at Station ALOHA (22.75˚N, 158˚W) in the North Pacific Subtropical Gyre (NPSG). Additionally, the spatial variability in these same properties was investigated along an east-west transect from California to Hawaii in the Fall of 2014. We used radiotracer techniques to determine the turnover times of the Pi or ATP pools respectively, and assessed the net production of dissolved organic phosphorus, and Pi hydrolysis rate from ATP. Pi concentrations in the upper water column at Station ALOHA are temporally highly dynamic, with periods of <10 nM-P to near 200 nM-P recorded within the top 50 m over the past decades of observations. During the California to Hawaii transect Pi concentrations showed a similarly large range (<10 to >200 nM-P), emphasizing the spatially and temporally mosaic nature of the upper ocean of this large biome. The Pi-pool turnover time ranged from a few hours to several weeks, and was strongly correlated with measured Pi pool concentrations (r2=0.8; n=30 Station ALOHA; n=15 transect). The calculated Pi uptake rates at Station ALOHA averaged 3.7±1.3 nM-P d-1 (n=30), reflecting the typically low maximum Pi uptake rates of the Prochlorococcus dominated community and the predominantly non-limiting Pi conditions. The Pi uptake rates along the transect were more variable than Station ALOHA (averaging 9.2±4.7 nM=P d-1, n=15), possibly due to a more diverse planktonic community structure, including stations with elevated concentrations of chlorophyll and primary productivity. The turnover time of the dissolved ATP pool was typically substantially shorter than for the Pi-pool (2-5 days at Station ALOHA; 0.3-2.5 days along the transect), likely reflecting its low nanomolar to picomolar ambient pool concentrations. However, at stations with the lowest SRP concentrations the

  4. Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP.

    PubMed

    Girardet, J L; Dupont, Y; Lacapere, J J

    1989-09-01

    Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.

  5. Strong Correlation Between Concentrations of Tenofovir (TFV) Emtricitabine (FTC) in Hair and TFV Diphosphate and FTC Triphosphate in Dried Blood Spots in the iPrEx Open Label Extension: Implications for Pre-exposure Prophylaxis Adherence Monitoring.

    PubMed

    Gandhi, Monica; Glidden, David V; Liu, Albert; Anderson, Peter L; Horng, Howard; Defechereux, Patricia; Guanira, Juan V; Grinsztejn, Beatriz; Chariyalertsak, Suwat; Bekker, Linda-Gail; Grant, Robert M

    2015-11-01

    Self-reported adherence to pre-exposure prophylaxis (PrEP) has limitations, raising interest in pharmacologic monitoring. Drug concentrations in hair and dried blood spots (DBS) are used to assess long-term-exposure; hair shipment/storage occurs at room temperature. The iPrEx Open Label Extension collected DBS routinely, with opt-in hair collection; concentrations were measured with liquid chromatography/tandem mass spectrometry. In 806 hair-DBS pairs, tenofovir (TFV) hair levels and TFV diphosphate (DP) in DBS were strongly correlated (Spearman coefficient r = 0.734; P < .001), as were hair TFV/DBS emtricitabine (FTC) triphosphate (TP) (r = 0.781; P < .001); hair FTC/DBS TFV-DP (r = 0.74; P < .001); hair FTC/DBS FTC-TP (r = 0.587; P < .001). Drug detectability was generally concordant by matrix. Hair TFV/FTC concentrations correlate strongly with DBS levels, which are predictive of PrEP outcomes.

  6. Phosphorothioate analogs of P1,P3-di(nucleosid-5'-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase.

    PubMed

    Kaczmarek, Renata; Krakowiak, Agnieszka; Korczyński, Dariusz; Baraniak, Janina; Nawrot, Barbara

    2016-11-01

    Di(nucleosid-5'-yl) polyphosphates (NPnN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g., HIT proteins). Their structural analogs may serve as molecular probes and potential therapeutic agents. Three P1,P3-bis-thio-analogs of symmetrical di(nucleosid-5'-yl) triphosphates (NP3N) bearing adenosine, guanosine or ribavirin residues (6, 7 and 8, respectively), were obtained by direct condensation of corresponding base-protected nucleoside-5'-O-(2-thio-1,3,2-oxathiaphospholane) with anhydrous phosphoric acid in the presence of DBU. Deprotected products 6 and 8 were separated into individual P-diastereoisomers, whereas 7 was partially separated to yield diastereomerically enriched fractions. The absolute configuration at P-stereogenic centers in the separated diastereoisomers was assigned by RP-HPLC analysis of the products of enzymatic digestion with snake venom phosphodiesterase. The Fhit-assisted hydrolysis rates for 6 and 7 are by 2-3 orders of magnitude lower than that for the reference AP3A, and depend on the configuration of the stereogenic phosphorus atoms, while 8 occurred to be resistant to this cleavage.

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

    SciTech Connect

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. )

    1991-01-01

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

  8. Inhibition of insulin release by synthetic peptides shows that the H3 region at the C-terminal domain of syntaxin-1 is crucial for Ca(2+)- but not for guanosine 5'-[gamma-thio]triphosphate-induced secretion.

    PubMed Central

    Martin, F; Salinas, E; Vazquez, J; Soria, B; Reig, J A

    1996-01-01

    Recently, we have described the presence and possible role of syntaxin in pancreatic beta-cells by using monoclonal antibodies [F. Martin, F. Moya, L. M. Gutierrez, J.A. Reig, B. Soria (1995) Diabetologia 38, 860-863]. In order to characterize further the importance of specific domains of this protein, the functional role of a particular region of the syntaxin-1 molecule has now been investigated by using two synthetic peptides, SynA and SynB, corresponding to two portions of the H3 region at the C-terminal domain of the protein, residues 229-251 and 197-219 respectively. Functional experiments carried out in permeabilized pancreatic beta-cells demonstrate that these peptides inhibit Ca(2+)-dependent insulin release in a dose-dependent manner. This effect is specific because peptides of the same composition but random sequence do not show the same effect. In contrast with this inhibitory effect on Ca(2+)-induced secretion, both peptides increase basal release. However, under the same conditions, SynA and SynB do not affect guanosine 5'-[gamma-thio]triphosphate-induced insulin release. These results demonstrate that specific portions of the H3 region of syntaxin-1 are involved in critical protein-protein interactions specifically during Ca(2+)-induced insulin secretion. PMID:8947488

  9. 4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

    PubMed

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

    2011-06-01

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

  10. Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling

    PubMed Central

    LI, WENKAI; WEI, SHENG; LIU, CHAOXU; SONG, MINGYU; WU, HUA; YANG, YONG

    2016-01-01

    An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process. PMID:26531757

  11. Polymerization of the triphosphates of AraC, 2',2'-difluorodeoxycytidine (dFdC) and OSI-7836 (T-araC) by human DNA polymerase alpha and DNA primase.

    PubMed

    Richardson, Katherine A; Vega, Tanya P; Richardson, Frank C; Moore, Chad L; Rohloff, John C; Tomkinson, Blake; Bendele, Raymond A; Kuchta, Robert D

    2004-12-15

    OSI-7836 (4'-thio-araC, T-araC) is a nucleoside analogue that shows efficacy against solid tumor xenograft models. We examined how the triphosphates of OSI-7836 (T-araCTP), cytarabine (araCTP), and gemcitabine (dFdCTP) affected the initiation of new DNA strands by the pol alpha primase complex. Whereas dFdCTP very weakly inhibited primase, both T-araCTP and araCTP potently inhibited this enzyme. Primase polymerized T-araCTP and araCTP more readily than its natural substrate, CTP, and incorporation resulted in strong chain termination. dFdCTP, araCTP, and T-araCTP inhibited pol alpha competitively with respect to dCTP. When exogenously added primentemplates were used, pol alpha incorporated all three analogues into DNA, and incorporation caused either weak chain termination (dFdCTP), strong termination (araCTP), or extremely strong termination (T-araC). Furthermore, pol alpha polymerized T-araCTP only nine-fold less well than dCTP, whereas it polymerized araCTP and dFdCTP 24- and 83-fold less well, respectively. The presence of these three analogues in the template strand resulted in significant pausing by pol alpha, although the site and severity of pausing varied between the analogues. During the elongation of primase-synthesized primers, a reaction that is thought to mimic the normal sequence of events during the initiation of new DNA strands, pol alpha polymerized all three compounds. However, incorporation of araCTP and dFdCTP resulted in minimal chain termination, while incorporation of T-araCTP still caused extremely strong termination. The implications of these results with respect to how these compounds affect cells are discussed.

  12. Oral administration of amino acidic supplements improves protein and energy profiles in skeletal muscle of aged rats: elongation of functional performance and acceleration of mitochondrial recovery in adenosine triphosphate after exhaustive exertion.

    PubMed

    Chen Scarabelli, Carol; McCauley, Roy B; Yuan, Zhaokan; Di Rezze, Justin; Patel, David; Putt, Jeff; Raddino, Riccardo; Allebban, Zuhair; Abboud, John; Scarabelli, Gabriele M; Chilukuri, Karuna; Gardin, Julius; Saravolatz, Louis; Faggian, Giuseppe; Mazzucco, Alessandro; Scarabelli, Tiziano M

    2008-06-02

    Sarcopenia is an inevitable age-related degenerative process chiefly characterized by decreased synthesis of muscle proteins and impaired mitochondrial function, leading to progressive loss of muscle mass. Here, we sought to probe whether long-term administration of oral amino acids (AAs) can increase protein and adenosine triphosphate (ATP) content in the gastrocnemius muscle of aged rats, enhancing functional performance. To this end, 6- and 24-month-old male Fisher 344 rats were divided into 3 groups: group A (6-month-old rats) and group B (24-month-old rats) were used as adult and senescent control group, respectively, while group C (24-month-old rats) was used as senescent treated group and underwent 1-month oral treatment with a mixture of mainly essential AAs. Untreated senescent animals exhibited a 30% reduction in total and fractional protein content, as well as a 50% reduction in ATP content and production, compared with adult control rats (p <0.001). Long-term supplementation with mixed AAs significantly improved protein and high-energy phosphate content, as well as the rate of mitochondrial ATP production, conforming their values to those of adult control animals (p <0.001). The improved availability of protein and high-energy substrates in the gastrocnemius muscle of treated aged rats paralleled a significant enhancement in functional performance assessed by swim test, with dramatic elongation of maximal exertion times compared with untreated senescent rats (p <0.001). In line with these findings, we observed that, after 6 hours of rest following exhaustive swimming, the recovery in mitochondrial ATP content was approximately 70% in adult control rats, approximately 60% in senescent control rats, and normalized in treated rats as compared with animals of the same age unexposed to maximal exertion (p <0.001). In conclusion, nutritional supplementation with oral AAs improved protein and energy profiles in the gastrocnemius of treated rats, enhancing

  13. A double-blind, randomized, comparative study of the use of a combination of uridine triphosphate trisodium, cytidine monophosphate disodium, and hydroxocobalamin, versus isolated treatment with hydroxocobalamin, in patients presenting with compressive neuralgias

    PubMed Central

    Goldberg, Henrique; Mibielli, Marco Antonio; Nunes, Carlos Pereira; Goldberg, Stephanie Wrobel; Buchman, Luiz; Mezitis, Spyros GE; Rzetelna, Helio; Oliveira, Lisa; Geller, Mauro; Wajnsztajn, Fernanda

    2017-01-01

    Context This paper reports on the results of treatment of compressive neuralgia using a combination of nucleotides (uridine triphosphate trisodium [UTP] and cytidine monophosphate disodium [CMP]) and vitamin B12. Objectives To assess the safety and efficacy of the combination of nucleotides (UTP and CMP) and vitamin B12 in patients presenting with neuralgia arising from neural compression associated with degenerative orthopedic alterations and trauma, and to compare these effects with isolated administration of vitamin B12. Methods A randomized, double-blind, controlled trial, consisting of a 30-day oral treatment period: Group A (n=200) receiving nucleotides + vitamin B12, and Group B (n=200) receiving vitamin B12 alone. The primary study endpoint was the percentage of subjects presenting pain visual analog scale (VAS) scores ≤20 at end of study treatment period. Secondary study endpoints included the percentage of subjects presenting improvement ≥5 points on the patient functionality questionnaire (PFQ); percentage of subjects presenting pain reduction (reduction in VAS scores at study end in relation to pretreatment); and number of subjects presenting adverse events. Results The results of this study showed a more expressive improvement in efficacy evaluations among subjects treated with the combination of nucleotides + vitamin B12, with a statistically significant superiority of the combination in pain reduction (evidenced by VAS scores). There were adverse events in both treatment groups, but these were transitory and no severe adverse event was recorded during the study period. Safety parameters were maintained throughout the study in both treatment groups. Conclusion The combination of uridine, cytidine, and vitamin B12 was safe and effective in the treatment of neuralgias arising from neural compression associated with degenerative orthopedic alterations and trauma. PMID:28243144

  14. 4-Alkyloxyimino Derivatives of Uridine-5′-triphosphate: Distal Modification of Potent Agonists as a Strategy for Molecular Probes of P2Y2, P2Y4, and P2Y6 Receptors

    PubMed Central

    2015-01-01

    Extended N4-(3-arylpropyl)oxy derivatives of uridine-5′-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N4-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N4-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N4-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs. PMID:24712832

  15. Cytosolic free Ca2+ oscillations induced by diadenosine 5',5"'-P1,P3-triphosphate and diadenosine 5',5"'-P1,P4-tetraphosphate in single rat hepatocytes are indistinguishable from those induced by ADP and ATP respectively.

    PubMed

    Green, A K; Cobbold, P H; Dixon, C J

    1995-09-01

    Diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) induce distinctive patterns of [Ca2+]i oscillations in single rat hepatocytes. We show here that [Ca2+]i oscillations induced by Ap3A and ADP are indistinguishable and that [Ca2+]i oscillations induced by Ap4A closely resemble those induced by ATP. These similarities embrace the following: (1) ADP and Ap3A invariably induce [Ca2+]i transients of short duration (approx. 9 s). Ap4A, like ATP, can induce, depending upon the individual cell, either transients of short duration (approx. 9 s), transients of much longer duration or a mixture of short and long transients within a single response. We show here that the pattern of oscillations induced by Ap4A is similar to that induced by ATP in the same hepatocyte. (2) Elevated intracellular cyclic AMP concentration modulates Ap3A-induced transients, like ADP-induced transients, through an increase in both the peak [Ca2+]i and the frequency of the transients. In contrast, Ap4A-induced transients, like ATP-induced transients, develop an increased duration or a sustained rise in [Ca2+]i, with no rise in peak [Ca2+]i. (3) Ap3A-induced transients, like ADP-induced transients, are abolished by low concentrations of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB; 5-10 nM), whereas long Ap4A-induced transients, like long ATP-induced transients, are refractory to high concentrations of PDB (100 nM). We propose that the [Ca2+]i oscillations induced in rat hepatocytes by Ap3A are mediated by the same purinoceptor that mediates the effects of ADP, whereas the oscillations induced by Ap4A are mediated by the same purinoceptor(s) that mediate the effects of ATP.

  16. 5'-triphosphate-siRNA activates RIG-I-dependent type I interferon production and enhances inhibition of hepatitis B virus replication in HepG2.2.15 cells.

    PubMed

    Chen, Xiaojuan; Qian, Yuanyu; Yan, Fei; Tu, Jian; Yang, Xingxing; Xing, Yaling; Chen, Zhongbin

    2013-12-05

    Hepatitis B virus (HBV) infection often results in acute or chronic viral hepatitis and other liver diseases including cirrhosis and hepatocellular carcinoma. Current therapies for HBV usually have severe side effects and can cause development of drug-resistant mutants. An alternative and safe immunotherapeutic approach for HBV infection is urgently needed for effective anti-HBV therapy. In this study, we propose a new strategy for anti-HBV therapy that activates type-I interferon (IFN) antiviral innate immunity through stimulating pattern-recognition receptors with RNA interference (RNAi) using a 5'-end triphosphate-modified small interfering RNA (3p-siRNA). We designed and generated a 3p-siRNA targeting overlapping region of S gene and P gene of the HBV genome at the 5'-end of pregenomic HBV RNA. Our results demonstrated that 3p-siRNA induced a RIG-I-dependent antiviral type-I IFN response when transfected into HepG2.2.15 cells that support HBV replication. The 3p-siRNA significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells in a RIG-I-dependent manner, and the antiviral effect of 3p-siRNA was superior to that of siRNA. Furthermore, 3p-siRNA had more pronounced inhibition effects on the replication of HBV DNA and the transcription of mRNA than that of siRNA. Finally, 3p-siRNA displayed antiviral activity with long-term suppression of HBV replication. In conclusion, our findings suggest that 3p-siRNA could act as a powerful bifunctional antiviral molecule with potential for developing a promising therapeutic against chronic HBV infection.

  17. Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling.

    PubMed

    Li, Wenkai; Wei, Sheng; Liu, Chaoxu; Song, Mingyu; Wu, Hua; Yang, Yong

    2016-01-01

    An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process.

  18. Rapid, quantitative determination of bacteria in water. [adenosine triphosphate

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Thomas, R. R.; Jeffers, E. L.; Deming, J. W. (Inventor)

    1978-01-01

    A bioluminescent assay for ATP in water borne bacteria is made by adding nitric acid to a water sample with concentrated bacteria to rupture the bacterial cells. The sample is diluted with sterile, deionized water, then mixed with a luciferase-luciferin mixture and the resulting light output of the bioluminescent reaction is measured and correlated with bacteria present. A standard and a blank also are presented so that the light output can be correlated to bacteria in the sample and system noise can be substracted from the readings. A chemiluminescent assay for iron porphyrins in water borne bacteria is made by adding luminol reagent to a water sample with concentrated bacteria and measuring the resulting light output of the chemiluminescent reaction.

  19. Bacterial adenosine triphosphate as a measure of urinary tract infection

    NASA Technical Reports Server (NTRS)

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

    1971-01-01

    Procedure detects and counts bacteria present in urine samples. Method also determines bacterial levels in other aqueous body fluids including lymph fluid, plasma, blood, spinal fluid, saliva and mucous.

  20. Interrelationship of islet metabolism, adenosine triphosphate content and insulin release

    PubMed Central

    Ashcroft, Stephen J. H.; Weerasinghe, L. Chatra C.; Randle, Philip J.

    1973-01-01

    The oxidation of some exogenous substrates and their effects on ATP content and insulin release in mouse pancreatic islets were measured. The ATP concentration of islets incubated without exogenous substrate shows a gradual decrease, which can be prevented by glucose or mannose (20mm) or leucine (2.5mm); d-glyceraldehyde (5mm) is as effective as glucose (5mm); fructose or N-acetylglucosamine (20mm), pyruvate (10mm) and dl-3-hydroxybutyrate (2mm) are less effective; galactose (20mm), acetate (10mm), octanoate (2mm) and succinate (10mm) have no ATP-maintaining ability. Islets oxidize glucose, mannose, glyceraldehyde, leucine and, less readily, N-acetylglucosamine and glucosamine; galactose, however, is poorly metabolized. Mannoheptulose inhibits the oxidation of glucose but not of glyceraldehyde. Insulin release, measured over a 2h incubation, is stimulated by glucose, mannose, leucine, glyceraldehyde or glucosamine but not by fructose or N-acetylglucosamine. The latter, however, potentiates the effects of glucose or glyceraldehyde (5mm) or leucine (2.5mm) on release; the potentiating effects are inhibited by mannoheptulose, which also blocks glucose-, but not glyceraldehyde- or leucine-stimulated release. In the presence of glucose (20mm), metabolic inhibitors depress insulin release and islet ATP content in parallel. However, rates of insulin release and ATP content measured after incubation with various combinations of exogenous substrates do not appear to be correlated. Sulphonylureas stimulate insulin release but decrease islet ATP concentrations. These results provide further evidence of a close association between the metabolic activity of exogenous substrates and their ability to initiate insulin release. Glucoreceptor models are formulated in the light of these observations and discussed. PMID:4199014

  1. Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.

    PubMed

    Draskovic, Petra; Saiardi, Adolfo; Bhandari, Rashna; Burton, Adam; Ilc, Gregor; Kovacevic, Miroslav; Snyder, Solomon H; Podobnik, Marjetka

    2008-03-01

    Eukaryotic cells produce a family of diverse inositol polyphosphates (IPs) containing pyrophosphate bonds. Inositol pyrophosphates have been linked to a wide range of cellular functions, and there is growing evidence that they act as second messengers. Inositol hexakisphosphate kinase (IP6K) is able to convert the natural substrates inositol pentakisphosphate (IP 5) and inositol hexakisphosphate (IP 6) to several products with an increasing number of phospho-anhydride bonds. In this study, we structurally analyzed IPs synthesized by three mammalian isoforms of IP6K from IP 5 and IP 6. The NMR and mass analyses showed a number of products with diverse, yet specific, stereochemistry, defined by the architecture of IP6K's active site. We now report that IP6K synthesizes both pyrophosphate (diphospho) as well as triphospho groups on the inositol ring. All three IP6K isoforms share the same activities both in vitro and in vivo.

  2. NTPDase2 and purinergic signaling control progenitor cell proliferation in neurogenic niches of the adult mouse brain.

    PubMed

    Gampe, Kristine; Stefani, Jennifer; Hammer, Klaus; Brendel, Peter; Pötzsch, Alexandra; Enikolopov, Grigori; Enjyoji, Keiichi; Acker-Palmer, Amparo; Robson, Simon C; Zimmermann, Herbert

    2015-01-01

    Nerve cells are continuously generated from stem cells in the adult mammalian subventricular zone (SVZ) and hippocampal dentate gyrus. We have previously noted that stem/progenitor cells in the SVZ and the subgranular layer (SGL) of the dentate gyrus express high levels of plasma membrane-bound nucleoside triphosphate diphosphohydrolase 2 (NTPDase2), an ectoenzyme that hydrolyzes extracellular nucleoside diphosphates and triphosphates. We inferred that deletion of NTPDase2 would increase local extracellular nucleoside triphosphate concentrations perturbing purinergic signaling and boosting progenitor cell proliferation and neurogenesis. Using newly generated mice globally null for Entpd2, we demonstrate that NTPDase2 is the major ectonucleotidase in these progenitor cell-rich areas. Using BrdU-labeling protocols, we have measured stem cell proliferation and determined long-term survival of cell progeny under basal conditions. Brains of Entpd2 null mice revealed increased progenitor cell proliferation in both the SVZ and the SGL. However, this occurred without noteworthy alterations in long-term progeny survival. The hippocampal stem cell pool and the pool of the intermediate progenitor type-2 cells clearly expanded. However, substantive proportions of these proliferating cells were lost during expansion at around type-3 stage. Cell loss was paralleled by decreases in cAMP response element-binding protein phosphorylation in the doublecortin-positive progenitor cell population and by an increase in labeling for activated caspase-3 levels. We propose that NTPDase2 has functionality in scavenging mitogenic extracellular nucleoside triphosphates in neurogenic niches of the adult brain, thereby acting as a homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion.

  3. Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

    PubMed Central

    Iandiev, Ianors; Wurm, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Robson, Simon C.; Zimmermann, Herbert

    2007-01-01

    Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y1 and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y1, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume. PMID:18404455

  4. Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5

    PubMed Central

    Vogiatzi, Fotini; Brandt, Dominique T.; Schneikert, Jean; Fuchs, Jeannette; Grikscheit, Katharina; Wanzel, Michael; Pavlakis, Evangelos; Charles, Joël P.; Timofeev, Oleg; Nist, Andrea; Mernberger, Marco; Kantelhardt, Eva J.; Siebolts, Udo; Bartel, Frank; Jacob, Ralf; Rath, Ariane; Moll, Roland; Grosse, Robert; Stiewe, Thorsten

    2016-01-01

    Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5′-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment. PMID:27956623

  5. Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay

    PubMed Central

    Lima, Juliana Maria; de Oliveira, Arthur Henrique Cavalcante

    2016-01-01

    The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave KM of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution. PMID:28070446

  6. Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay.

    PubMed

    Calil, Felipe Antunes; Lima, Juliana Maria; de Oliveira, Arthur Henrique Cavalcante; Mariotini-Moura, Christiane; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lucia

    2016-01-01

    The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave KM of 0.317 ± 0.044 mmol·L(-1), which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L(-1), which is in accordance with the data for the enzyme in solution.

  7. Enzymes that hydrolyze adenine nucleotides in platelets and polymorphisms in the alpha2 gene of integrin alpha2beta1 in patients with von Willebrand disease.

    PubMed

    Santos, Karen Freitas; Battisti, Vanessa; Corrêa, Maísa de Carvalho; Mann, Thaís Rapachi; Pereira, Renata da Silva; Araújo, Maria do Carmo; Brülê, Alice Odete; Schetinger, Maria Rosa Chitolina; Morsch, Vera Maria

    2010-07-01

    Von Willebrand disease (VWD) is one of the most common inherited bleeding diseases caused by a qualitative or quantitative deficiency of the von Willebrand factor (FvW). FvW is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells and it is present in the subendothelial matrix, blood plasma, platelets, and endothelium. This glycoprotein plays an important role in thrombus formation by initiating platelet adhesion to sites of injury as well as platelet aggregation. The aim of this study was to evaluate the activities of enzymes that hydrolyze adenine nucleotides in platelets, ristocetin-induced platelet aggregation (RIPA), and polymorphisms of the alpha2 gene of alpha2beta1 integrin from VWD patients. Platelet nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activities were verified in 14 VWD patients. For RIPA determination, a final concentration of 1.25 mg/ml of ristocetin was used. Polymorphisms of the alpha2 gene were analyzed through PCR. Platelet NTPDase and E-NPP were decreased in VWD patients. 5'-Nucleotidase activity was not statistically significant between controls and VWD patients. RIPA was significantly reduced, with an allelic frequency of 78.57% for 807C in VWD patients. Our results indicated reduced platelet NTPDase and E-NPP activities which might be related to the low platelet adhesiveness. The prevalence of the 807C allele might account for the variability in bleeding in VWD.

  8. Heterogeneity in extracellular nucleotide hydrolysis among clinical isolates of Trichomonas vaginalis

    PubMed Central

    TASCA, T.; BONAN, C. D.; DE CARLI, G. A.; SARKIS, J.J.F.; ALDERETE, J. F.

    2007-01-01

    SUMMARY Trichomonas vaginalis is a parasitic protozoan that causes trichomonosis, a sexually-transmitted disease, with serious sequelae to women and men. As the host-parasite relationship is complex, it is important to investigate biochemical aspects of the parasite that contribute to our understanding of trichomonal biology and pathogenesis. Nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1), which hydrolyses extracellular ATP and ADP, and ecto-5′-nucleotidase, which hyrolyses AMP, have been characterized in laboratory isolates of T. vaginalis. Here we show that the extracellular ATP:ADP hydrolysis ratio varies among fresh clinical isolates, which presented higher ATPase and ADPase activities than long-term-grown isolates. Growth of parasites in iron-replete and iron-depleted medium resulted in different, albeit minor, patterns in extracellular ATP and ADP hydrolysis among isolates. Importantly, some isolates had low or absent ecto-5′-nucleotidase activity, regardless of environmental conditions tested. For isolates with ecto-5′-nucleotidase activity, high- and low-iron trichomonads had increased and decreased levels of activity, respectively, compared to organisms grown in normal TYM-serum medium. This suggests a regulation in expression of either the enzyme amounts and/or activity under the control of iron. Finally, we found no correlation between the presence or absence of dsRNA virus infection among trichomonad isolates and NTPDase and ecto-5′-nucleotidase activities. PMID:16038398

  9. Biochemical characterization of Arabidopsis APYRASE family reveals their roles in regulating endomembrane NDP/NMP homoeostasis.

    PubMed

    Chiu, Tsan-Yu; Lao, Jeemeng; Manalansan, Bianca; Loqué, Dominique; Roux, Stanley J; Heazlewood, Joshua L

    2015-11-15

    Plant apyrases are nucleoside triphosphate (NTP) diphosphohydrolases (NTPDases) and have been implicated in an array of functions within the plant including the regulation of extracellular ATP. Arabidopsis encodes a family of seven membrane bound apyrases (AtAPY1-7) that comprise three distinct clades, all of which contain the five conserved apyrase domains. With the exception of AtAPY1 and AtAPY2, the biochemical and the sub-cellular characterization of the other members are currently unavailable. In this research, we have shown all seven Arabidopsis apyrases localize to internal membranes comprising the cis-Golgi, endoplasmic reticulum (ER) and endosome, indicating an endo-apyrase classification for the entire family. In addition, all members, with the exception of AtAPY7, can function as endo-apyrases by complementing a yeast double mutant (Δynd1Δgda1) which lacks apyrase activity. Interestingly, complementation of the mutant yeast using well characterized human apyrases could only be accomplished by using a functional ER endo-apyrase (NTPDase6), but not the ecto-apyrase (NTPDase1). Furthermore, the substrate specificity analysis for the Arabidopsis apyrases AtAPY1-6 indicated that each member has a distinct set of preferred substrates covering various NDPs (nucleoside diphosphates) and NTPs. Combining the biochemical analysis and sub-cellular localization of the Arabidopsis apyrases family, the data suggest their possible roles in regulating endomembrane NDP/NMP (nucleoside monophosphate) homoeostasis.

  10. CD39 modulates endothelial cell activation and apoptosis.

    PubMed Central

    Goepfert, C.; Imai, M.; Brouard, S.; Csizmadia, E.; Kaczmarek, E.; Robson, S. C.

    2000-01-01

    BACKGROUND: CD39 is the dominant vascular nucleoside triphosphate diphosphohydrolase (NTPDase) that exerts major effects on platelet reactivity by the regulated hydrolysis of extracellular adenine nucleotides. The effects of NTPDases on endothelial cell (EC) activation and apoptosis remain unexplored. MATERIAL AND METHODS: Recombinant replication-deficient adenoviruses were constructed with human CD39 cDNA (rAdCD39) or the bacterial beta-galactosidase (rAdbetagal). RESULTS: Intact human umbilical vein EC cultures infected with rAdCD39 had substantial and stable increases in NTPDase biochemical activity (14.50 +/- 3.50 Pi nmole/well/min), when contrasted with noninfected cells (0.95 +/- 0.002) and rAdbetagal infected cells (1.01 +/- 0.02; p<0.005). Increased NTPDase activity efficiently inhibited immediate type 2Y purinergic receptor (P2Y)-mediated EC activation responses viz. von Willebrand factor secretion in response to extracellular ATP. In addition, CD39 up-regulation blocked ATP-induced translocation of the transcription nuclear factor (NF)-kappaB to the cell nucleus, and abrogated transcription of mRNA encoding E-selectin, and consequent protein synthesis. CD39 also decreased the extent of apoptosis triggered by putative type-2X purinergic (P2X7) receptors in response to high concentrations of extracellular ATP in vitro. CONCLUSION: These properties of CD39 indicate primary vascular protective effects with potential therapeutic applications. PMID:10997340

  11. Breakthroughs spotlighting roles for extracellular nucleotides and apyrases in stress responses and growth and development.

    PubMed

    Clark, Greg B; Morgan, Reginald O; Fernandez, Maria-Pilar; Salmi, Mari L; Roux, Stanley J

    2014-08-01

    Animal and plant cells release nucleotides into their extracellular matrix when touched, wounded, and when their plasma membranes are stretched during delivery of secretory vesicles and growth. These released nucleotides then function as signaling agents that induce rapid increases in the concentration of cytosolic calcium, nitric oxide and superoxide. These, in turn, are transduced into downstream physiological changes. These changes in plants include changes in the growth of diverse tissues, in gravitropism, and in the opening and closing of stomates. The concentration of extracellular nucleotides is controlled by various phosphatases, prominent among which are apyrases EC 3.6.1.5 (nucleoside triphosphate diphosphohydrolases, NTPDases). This review provides phylogenetic and pHMM analyses of plant apyrases as well as analysis of predicted post-translational modifications for Arabidopsis apyrases. This review also summarizes and discusses recent advances in research on the roles of apyrases and extracellular nucleotides in controlling plant growth and development. These include new findings that document how apyrases and extracellular nucleotides control auxin transport, modulate stomatal aperture, and mediate biotic and abiotic stress responses, and on how apyrase suppression leads to growth inhibition.

  12. Resveratrol triggers ER stress-mediated apoptosis by disrupting N-linked glycosylation of proteins in ovarian cancer cells.

    PubMed

    Gwak, HyeRan; Kim, Soochi; Dhanasekaran, Danny N; Song, Yong Sang

    2016-02-28

    Malignant tumors have a high glucose demand and alter cellular metabolism to survive. Herein, focusing on the utility of glucose metabolism as a therapeutic target, we found that resveratrol induced endoplasmic reticulum (ER) stress-mediated apoptosis by interrupting protein glycosylation in a cancer-specific manner. Our results indicated that resveratrol suppressed the hexosamine biosynthetic pathway and interrupted protein glycosylation through GSK3β activation. Application of either biochemical intermediates of the hexosamine pathway or small molecular inhibitors of GSK3β reversed the effects of resveratrol on the disruption of protein glycosylation. Additionally, an ER UDPase, ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), modulated protein glycosylation by Akt attenuation in response to resveratrol. By inhibition or overexpression of Akt functions, we confirmed that the glycosylation activities were dependent on ENTPD5 expression and regulated by the action of Akt in ovarian cancer cells. Resveratrol-mediated disruption of protein glycosylation induced cellular apoptosis as indicated by the up-regulation of GADD153, followed by the activation of ER-stress sensors (PERK and ATF6α). Thus, our results provide novel insight into cancer cell metabolism and protein glycosylation as a therapeutic target for cancers.

  13. Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism

    PubMed Central

    Pluskota, Elzbieta; Ma, Yi; Bledzka, Kamila M.; Bialkowska, Katarzyna; Soloviev, Dmitry A.; Szpak, Dorota; Podrez, Eugene A.; Fox, Paul L.; Hazen, Stanley L.; Dowling, James J.; Ma, Yan-Qing

    2013-01-01

    Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2+/− mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2+/− mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2+/− mice, kindlin-2+/− endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5′-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5′-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73) were increased twofold to threefold on kindlin-2+/− ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2+/− mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis. PMID:23896409

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

    PubMed Central

    Bracco, Paula Andreghetto; Bertoni, Ana Paula Santin; Wink, Márcia Rosângela

    2014-01-01

    The protooncogene PCPH was recently identified as being the ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5). This protooncogene is converted into an oncogene by a single base pair deletion, resulting in frame change and producing a premature stop codon, leading to a mutated protein (mt-PCPH) with only 27 kDa, which is much smaller than the original 47 kDa protein. Overexpression of the PCPH as well as the mutated PCPH increases the cellular resistance to stress and apoptosis. This is intriguing considering that the active form, that is, the oncogene, is the mutated PCPH. Several studies analyzed the expression of NTPDase5/mt-PCPH in a wide range of tumor cells and evaluated its role and mechanisms in cancer and other pathogenic processes. The main point of this review is to integrate the findings and proposed theories about the role played by NTPDase5/mt-PCPH in cancer progression, considering that these proteins have been suggested as potential early diagnostic tools and therapy targets. PMID:25045656

  15. Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

    PubMed

    Porowińska, Dorota; Czarnecka, Joanna; Komoszyński, Michał

    2014-07-01

    NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

  16. Ectonucleotidase CD39-driven control of postinfarction myocardial repair and rupture

    PubMed Central

    Sutton, Nadia R.; Hayasaki, Takanori; Hyman, Matthew C.; Anyanwu, Anuli C.; Liao, Hui; Petrovic-Djergovic, Danica; Badri, Linda; Baek, Amy E.; Walker, Natalie; Fukase, Keigo; Visovatti, Scott H.; Horste, Ellen L.; Ray, Jessica J.; Goonewardena, Sascha N.

    2017-01-01

    Mechanical complications of myocardial infarction (MI) are often fatal. Little is known about endogenous factors that predispose to myocardial rupture after MI. Ectonucleoside triphosphate diphosphohydrolase (CD39) could be a critical mediator of propensity to myocardial rupture after MI due to its role in modulating inflammation and thrombosis. Using a model of permanent coronary artery ligation, rupture was virtually abrogated in cd39–/– mice versus cd39+/+ controls, with elevated fibrin and collagen deposition and marked neutrophil and macrophage influx. Macrophages were found to display increased surface expression of CD301 and CD206, marking a reparative phenotype, driven by increased extracellular ATP and IL-4 in the infarcted myocardium of cd39–/– mice. A myeloid-specific CD39-knockout mouse also demonstrated protection from rupture, with an attenuated rupture phenotype, suggesting that complete ablation of CD39 provides the greatest degree of protection in this model. Absence of CD39, either globally or in a myeloid lineage–restricted fashion, skews the phenotype toward alternatively activated (reparative) macrophage infiltration following MI. These studies reveal a previously unrecognized and unexpected role of endogenous CD39 to skew macrophage phenotype and promote a propensity to myocardial rupture after MI. PMID:28097233

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

    PubMed

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

    2013-06-01

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

  18. High expression of CD39 in gastric cancer reduces patient outcome following radical resection

    PubMed Central

    Cai, Xiao-Yan; Wang, Xue-Fei; Li, Jun; Dong, Jiang-Nan; Liu, Jiang-Qi; Li, Neng-Ping; Yun, Bei; Xia, Rong-Long; Qin, Jing; Sun, Yi-Hong

    2016-01-01

    Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also known as cluster of differentiation (CD)39, is the rate-limiting enzyme in the generation of immunosuppressive adenosine and is important in tumor progression. The present study evaluated the expression of CD39+ and CD39+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells in gastric cancer (GC), and determined their prognostic roles in patients with GC following radical resection. It was observed that CD39 was expressed at significantly higher rates in tumor tissues as compared with paired peritumoral tissues. Overexpression of tumor CD39 was correlated with overall survival (OS). Furthermore, CD39 expression in GC tissues exhibited a prognostic role in OS. The CD39+ FoxP3+/FoxP3+ ratio in tumor tissues was higher than that in paired peritumoral tissues, and CD39+ FoxP3+ Treg cells were a better prognostic indicator than FoxP3+ Treg cells for OS. Collectively, our study indicates that overexpression of CD39 in GC is a predictor of poor outcome for GC patients following radical resection. CD39+ FoxP3+ Treg cells are a potential target for cancer immunotherapy. PMID:27895775

  19. Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5.

    PubMed

    Vogiatzi, Fotini; Brandt, Dominique T; Schneikert, Jean; Fuchs, Jeannette; Grikscheit, Katharina; Wanzel, Michael; Pavlakis, Evangelos; Charles, Joël P; Timofeev, Oleg; Nist, Andrea; Mernberger, Marco; Kantelhardt, Eva J; Siebolts, Udo; Bartel, Frank; Jacob, Ralf; Rath, Ariane; Moll, Roland; Grosse, Robert; Stiewe, Thorsten

    2016-12-27

    Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5'-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.

  20. Characterization of E-NTPDase (EC 3.6.1.5) activity in hepatic lymphocytes: A different activity profile from peripheral lymphocytes.

    PubMed

    Doleski, Pedro H; Adefegha, Stephen A; Cabral, Fernanda L; Leal, Daniela B R

    2017-03-01

    The activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E-NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E-NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca(2+) . In addition, in the presence of specific E-NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E-ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03μM and 214.40 ± 0.06μM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 ƞmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E-NTPDase. The higher E-NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL.

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

    PubMed

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

    1984-05-22

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

  2. Rapid Phytochrome-mediated Changes in Adenosine 5'-Triphosphate Content of Etiolated Bean Buds.

    PubMed

    White, J M; Pike, C S

    1974-01-01

    This study was designed to determine the effects of red and far red irradiation on ATP metabolism in etiolated bean buds (Phaseolus vulgaris L. var. Red Kidney). Compared to dark controls, red irradiated buds show an initial decline in ATP content at 15 seconds following a 5-minute irradiation. ATP content then rapidly rises to a peak at 1 minute, and then slowly returns to the baseline. The 1-minute promotion of ATP content is red/far red reversible. Acetylcholine does not appear to mimic red light in this system; it causes a marked decrease in ATP content.

  3. Elevated Levels of Plasma Phenylalanine in Schizophrenia: A Guanosine Triphosphate Cyclohydrolase-1 Metabolic Pathway Abnormality?

    PubMed Central

    Okusaga, Olaoluwa; Muravitskaja, Olesja; Fuchs, Dietmar; Ashraf, Ayesha; Hinman, Sarah; Giegling, Ina; Hartmann, Annette M.; Konte, Bettina; Friedl, Marion; Schiffman, Jason; Hong, Elliot; Reeves, Gloria; Groer, Maureen; Dantzer, Robert

    2014-01-01

    Background Phenylalanine and tyrosine are precursor amino acids required for the synthesis of dopamine, the main neurotransmitter implicated in the neurobiology of schizophrenia. Inflammation, increasingly implicated in schizophrenia, can impair the function of the enzyme Phenylalanine hydroxylase (PAH; which catalyzes the conversion of phenylalanine to tyrosine) and thus lead to elevated phenylalanine levels and reduced tyrosine levels. This study aimed to compare phenylalanine, tyrosine, and their ratio (a proxy for PAH function) in a relatively large sample of schizophrenia patients and healthy controls. Methods We measured non-fasting plasma phenylalanine and tyrosine in 950 schizophrenia patients and 1000 healthy controls. We carried out multivariate analyses to compare log transformed phenylalanine, tyrosine, and phenylalanine:tyrosine ratio between patients and controls. Results Compared to controls, schizophrenia patients had higher phenylalanine (p<0.0001) and phenylalanine: tyrosine ratio (p<0.0001) but tyrosine did not differ between the two groups (p = 0.596). Conclusions Elevated phenylalanine and phenylalanine:tyrosine ratio in the blood of schizophrenia patients have to be replicated in longitudinal studies. The results may relate to an abnormal PAH function in schizophrenia that could become a target for novel preventative and interventional approaches. PMID:24465804

  4. Nucleotide triphosphates are required for the transport of glycolate oxidase into peroxisomes.

    PubMed

    Brickner, D G; Olsen, L J

    1998-01-01

    All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

  5. Sperm morphology, adenosine triphosphate (ATP) concentration and swimming velocity: unexpected relationships in a passerine bird

    PubMed Central

    Bennison, Clair; Brookes, Lola; Slate, Jon; Birkhead, Tim

    2016-01-01

    The relationship between sperm energetics and sperm function is poorly known, but is central to our understanding of the evolution of sperm traits. The aim of this study was to examine how sperm morphology and ATP content affect sperm swimming velocity in the zebra finch Taeniopygia guttata. We exploited the high inter-male variation in this species and created extra experimental power by increasing the number of individuals with very long or short sperm through artificial selection. We found a pronounced quadratic relationship between total sperm length and swimming velocity, with velocity increasing with length up to a point, but declining in the very longest sperm. We also found an unexpected negative association between midpiece length and ATP content: sperm with a short midpiece generally contained the highest concentration of ATP. Low intracellular ATP is therefore unlikely to explain reduced swimming velocity among the very longest sperm (which tend to have a shorter midpiece). PMID:27559067

  6. Adenosine triphosphate (ATP) reduces amyloid-β protein misfolding in vitro.

    PubMed

    Coskuner, Orkid; Murray, Ian V J

    2014-01-01

    Alzheimer's disease (AD) is a devastating disease of aging that initiates decades prior to clinical manifestation and represents an impending epidemic. Two early features of AD are metabolic dysfunction and changes in amyloid-β protein (Aβ) levels. Since levels of ATP decrease over the course of the disease and Aβ is an early biomarker of AD, we sought to uncover novel linkages between the two. First and remarkably, a GxxxG motif is common between both Aβ (oligomerization motif) and nucleotide binding proteins (Rossmann fold). Second, ATP was demonstrated to protect against Aβ mediated cytotoxicity. Last, there is structural similarity between ATP and amyloid binding/inhibitory compounds such as ThioT, melatonin, and indoles. Thus, we investigated whether ATP alters misfolding of the pathologically relevant Aβ42. To test this hypothesis, we performed computational and biochemical studies. Our computational studies demonstrate that ATP interacts strongly with Tyr10 and Ser26 of Aβ fibrils in solution. Experimentally, both ATP and ADP reduced Aβ misfolding at physiological intracellular concentrations, with thresholds at ~500 μM and 1 mM respectively. This inhibition of Aβ misfolding is specific; requiring Tyr10 of Aβ and is enhanced by magnesium. Last, cerebrospinal fluid ATP levels are in the nanomolar range and decreased with AD pathology. This initial and novel finding regarding the ATP interaction with Aβ and reduction of Aβ misfolding has potential significance to the AD field. It provides an underlying mechanism for published links between metabolic dysfunction and AD. It also suggests a potential role of ATP in AD pathology, as the occurrence of misfolded extracellular Aβ mirrors lowered extracellular ATP levels. Last, the findings suggest that Aβ conformation change may be a sensor of metabolic dysfunction.

  7. Adenosine triphosphate depletion reverses sodium-dependent, neuronal uptake of glutamate in rat hippocampal slices.

    PubMed

    Madl, J E; Burgesser, K

    1993-10-01

    Extracellular accumulations of excitatory amino acids (EAAs) may mediate ischemic neuronal damage. Metabolic insults can decrease Na+ and K+ plasma membrane gradients, thereby reducing the driving force for uptake of EAAs into cells by Na(+)-dependent EAA cotransporters. EAA accumulations could result from decreased uptake and increased release due to reversal of these cotransporters. ATP depletion, uptake, and release of EAAs were measured by HPLC in slices treated with metabolic inhibitors. Inhibition and reversal of cotransporters were determined by uptake or release of D,L-threo-beta-hydroxyaspartate (OH-Asp), an EAA analog with high affinity for cotransporters. Moderate ATP depletion (7 > ATP nmol/mg protein > 3) reduced uptake by cotransporters without increasing release of EAAs. When ATP was severely depleted (ATP < 2 nmol/mg protein), increased release of EAAs and preloaded OH-Asp occurred, consistent with reversal of cotransporters. Release of glutamine and asparagine was not increased, confirming that release was not primarily due to nonselective increased membrane permeability. ATP depletion and ouabain acted synergistically to produce EAA release, strongly suggesting release was largely mediated by inhibition of Na/K-ATPases. Severe ATP depletion decreased glutamate-like immunoreactivity primarily in axonal terminal-like structures, suggesting release occurred primarily from terminals. Moderate ATP depletion may increase extracellular EAAs by decreasing uptake. Severe ATP depletion may further increase EAAs by reversing uptake, thereby releasing cytosolic neuronal pools of EAAs.

  8. Monitoring of bacterial contamination of dental unit water lines using adenosine triphosphate bioluminescence.

    PubMed

    Watanabe, A; Tamaki, N; Yokota, K; Matsuyama, M; Kokeguchi, S

    2016-12-01

    Bacterial contamination of dental unit waterlines (DUWLs) was evaluated using ATP bioluminescence analysis and a conventional culture method. Water samples (N=44) from DUWLs were investigated for heterotrophic bacteria by culture on R2A agar, which gave counts ranging from 1.4×10(3) to 2.7×10(5) cfu/mL. The ATP bioluminescence results for DUWL samples ranged from 6 to 1189 relative light units and could be obtained within 1min; these correlated well with the culture results (r=0.727-0.855). We conclude that the results of the ATP bioluminescence assay accurately reflect the results of conventional culture-based testing. This method is potentially useful for rapid and simple monitoring of DUWL bacterial contamination.

  9. Ultrasensitive bioluminescent determinations of adenosine triphosphate (ATP) for investigating the energetics of host-grown microbes

    NASA Technical Reports Server (NTRS)

    Hanks, J. H.; Dhople, A. M.

    1975-01-01

    Stability and optimal concentrations of reagents were studied in bioluminescence assay of ATP levels. Luciferase enzyme was prepared and purified using Sephadex G-100. Interdependencies between enzyme and luciferin concentrations in presence of optimal Mg are illustrated. Optimal ionic strength was confirmed to be 0.05 M for the four buffers tested. Adapted features of the R- and H-systems are summarized, as well as the percentages of ATP pools released from representative microbes by heat and chloroform.

  10. Appearance of adenosine triphosphate in the perfusate from working frog heart.

    PubMed

    Doyle, T B; Forrester, T

    1985-09-01

    Frog hearts (Rana pipiens) were perfused in situ with Ringer's solution and the perfusate tested on firefly extract for the presence of ATP. At a normal perfusion pressure of 8 cm. H2O the rate of release of ATP into the perfusate was 8.8 (+/- S.E.1.7) pmoles.min-1. When the workload was increased by raising the perfusion pressure to 12 cm. H2O the rate of release increased to 28.3 (+/- S.E.4.8) pmoles.min-1. The rate of release was found to be proportional to the amount of workload imposed upon the heart. It is postulated that the trigger for release is hypoxia and that the release of ATP from the cardiac cell will augment contractility of the myocardium through its action upon adjacent cells via the P2 purinergic receptor. cells via the P2 purinergic receptor.

  11. A Genetic Analysis of the Pteridine Biosynthetic Enzyme, Guanosine Triphosphate Cyclohydrolase, in DROSOPHILA MELANOGASTER

    PubMed Central

    Mackay, William J.; O'Donnell, Janis M.

    1983-01-01

    Strains with mutant eye color were surveyed for levels of GTP cyclohydrolase (GTP CH), the first enzyme acting in the biosynthesis of pteridines, the pigments causing red eye color in Drosophila. Six strains were found to have reduced GTP CH activity. In five of the six strains, the reduction of activity is apparent only in the adult head of homozygous mutants. We show that mutations in Punch (2-97, Pu) have severe effects on GTP CH activity. In most cases, the reduction of activity is apparent in all tissues and stages that express the enzyme. The activity of GTP CH is shown to be closely correlated with the number of Pu+ genes in the genome. One ethyl methanesulfonate (EMS)-induced Pu mutant has a GTP CH enzyme that is unstable when compared with the wild-type enzyme. Mutations in Pu fall into three general classes. The largest class has a recessive lethal and eye color phenotype, 50% or higher GTP CH activity in heterozygotes, and equivalent defects in all tissues. A second class is dominant in eye color phenotype and recessive lethal, with less than 50% GTP CH activity in heterozygotes. The third class is homozygous viable and has severe reduction of activity in the adult head, but no or less severe loss in other tissues. PMID:6413298

  12. Herpes simplex virus 1 primase employs watson-crick hydrogen bonding to identify cognate nucleoside triphosphates.

    PubMed

    Ramirez-Aguilar, Kathryn A; Moore, Chad L; Kuchta, Robert D

    2005-11-29

    We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.

  13. Some aspects of adenosine triphosphate synthesis from adenine and adenosine in human red blood cells

    PubMed Central

    Whittam, R.; Wiley, J. S.

    1968-01-01

    1. The synthesis of ATP has been studied in human erythrocytes. Fresh cells showed no net synthesis of ATP when incubated with adenine or adenosine, although labelled adenine was incorporated into ATP in small amounts. 2. Cold-stored cells (3-6 weeks old) became progressively depleted of adenine nucleotides but incubation with adenosine or adenine plus inosine restored the ATP concentration to normal within 4 hr. Incorporation of labelled adenine or adenosine into the ATP of incubated stored cells corresponded to net ATP synthesis by these cells. 3. Synthesis of ATP from adenosine plus adenine together was 75% derived from adenine and only 25% from adenosine, indicating that nucleotide synthesis from adenine inhibits the simultaneous synthesis of nucleotide from adenosine. PMID:5723519

  14. Cytidine diphosphate choline administration activates brain cytidine triphosphate: phosphocholine cytidylytransferase in aged rats.

    PubMed

    Giménez, R; Soler, S; Aguilar, J

    1999-10-08

    Beneficial effects of cytidine (5') diphosphocholine (CDP-choline) administration on several diseases including brain aging, ischemia and stroke are based on an increase in membrane phospholipid turnover. We have studied the possible involvement of CTP:phosphocholine cytidylyltransferase (CT) in this mechanism by measuring its gene expression and enzyme activity in the brains of young and aged rats treated with 500 mg/kg per day of CDP-choline. Older animals showed higher (57%) of total CT activity in particulate (active) fraction than younger animals (46%). Treatment of aged animals for 8, 16, or 60 days had no effect on the CT gene expression but increased activation of the CT by translocation to membranes. The particulate fraction rose from 57% of total activity to more than 65% after 2 months of treatment. This may explain the long-term repairing effects of CDP-choline on damaged membranes of aged animals.

  15. A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. The mode of action of the enzyme.

    PubMed

    Anai, M; Yamanaka, M; Shibata, T; Mihara, T; Nishimoto, T

    1975-07-01

    The acid-soluble products of exhaustive digestion of native DNA with Bacillus laterosporus DNase consist of 6.5% of mononucleotides and 93.5% of oligonucleotides with an average chain length of 3.2. The results of viscometric studies and inactivation of transforming DNA indicate the existence of acid-insoluble intermediates and the selective degradation of the population of substrate molecules rather than a random nucleolytic action. Furthermore, sucrose density gradient analysis of partially digested DNA showed that the initial DNA added as a substrate disappeared progressively during the reaction, being replaced by much more slowly sedimenting acid-insoluble materials, which were eventually degraded into acid-soluble end products during the reaction; products intermediate in size between these two components were not detectable. Studies with DNA labeled at the 3'-terminus indicate that Bacillus laterosporus DNase does not attack DNA from 3'-hydroxyl ends to yeild acid-soluble or acid-insoluble materials in a random manner. The results presented in this paper indicate that the nature of the attack of B. laterosporus nuclease is similar to that previously proposed for Micrococcus luteus DNase. The possibility of the sequential release of acid-insoluble intermediate fragments as well as acid-soluble products from the terminal portion of DNA by the enzyme is discussed.

  16. Two pH optima of adenosine 5'-triphosphate dependent deoxyribonuclease from Bacillus laterosporus.

    PubMed

    Fujiyoshi, T; Nakayama, J; Anai, M

    1982-08-17

    The various catalytic activities of the ATP-dependent deoxyribonuclease (DNase) of Bacillus laterosporus have pH optima at 6.3 and 8.3. Although the pH profile of ATP-dependent DNase activity on duplex DNA is bell shaped with a maximum at about pH 8.3, ATP-dependent DNAse activity on single-stranded DNA has optima at pH 6.3 and 8.3. ATPase activities dependent on double-stranded and single-stranded DNA have a high bell-shaped peak with a maximum at pH 6.3 with a low and broad shoulder at about pH 8.3. ATP-independent DNase activity also has optima at pH 6.3 and 8.3. The ratio of the amount of ATP hydrolyzed per number of cleaved phosphodiester bonds in DNA increases with decrease in the pH value of the reaction. The ratios obtained at pH 8.3 and 6.3 were respectively about 3 and 22 with duplex DNA as substrate and 5 and 17 with single-stranded DNA as substrate. Formation of a single-stranded region of 15000-20000 nucleotides, which is linked to duplex DNA and about half of which has 3'-hydroxyl termini, was observed at about pH 6.3, but not at above pH 7.5. Furthermore, the optimum concentrations of divalent cations for the activity producing the single-stranded region and the activity hydrolyzing ATP were identical (3 mM Mn2+ or 5 mM Mg2+). Thus the two activities are closely related. These results indicate that the enzyme has two different modes of action on duplex DNA which are modulated by the pH.

  17. An adenosine triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Improved purification, subunit structure and substrate specificity.

    PubMed

    Fujiyoshi, T; Anai, M

    1981-04-01

    The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.

  18. Extracellular adenosine triphosphate activates calcium mobilization in human phagocytic leukocytes and neutrophil/monocyte progenitor cells.

    PubMed Central

    Cowen, D S; Lazarus, H M; Shurin, S B; Stoll, S E; Dubyak, G R

    1989-01-01

    We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation. The average cytosolic [Ca2+] in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic [Ca2+] in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods. Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny. Significant ATP-induced increases in cytosolic [Ca2+] were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes. Extensive characterization of the ATP-induced changes in [Ca2+] observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors. Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes. PMID:2708526

  19. Preparation and characterization of salmon calcitonin-sodium triphosphate ionic complex for oral delivery.

    PubMed

    Lee, Hea Eun; Lee, Min Jung; Park, Cho Rong; Kim, A Young; Chun, Kyung Hwa; Hwang, Hee Jin; Oh, Dong Ho; Jeon, Sang Ok; Kang, Jae Seon; Jung, Tae Sung; Choi, Guang Jin; Lee, Sangkil

    2010-04-19

    Even though salmon calcitonin (sCT) has been known as a potent hypocalcemic agent, only injection or nasal spray products are available on the market. In order to develop oral delivery system of the agent, a novel sCT-sodium tripolyphosphate (STPP) ionic complex was fabricated and also characterized. For the optimization of the ionic complexation, the effect of incubation time and molar ratio between sCT and STPP was evaluated. Particle size of the ionic complex in aqueous media, SEM images, DSC, FT-IR, in vitro release test, stability within the simulated intestinal fluid, and hypocalcemic effect were evaluated. The optimal molar complexation ratio of sCT to STPP was ranged from 1:5 to 1:10 and the complexation efficiency was about 95%. The SEM image has shown that the freeze dried ionic complex has rough morphology in their surface and the particle size in PBS (pH 7.4) was about 220nm. The DSC and FT-IR results provided evidences for ionic interaction between -NH(2) groups and -P horizontal lineO groups of sCT and STPP, respectively. The sCT ionic complex has shown sustained sCT releasing characteristics for 3weeks. The sCT-STPP ionic complex was protective to enzymatic attack and in vivo animal data revealed that the present ionic complex would show continuous hypocalcemic effect. Conclusively, the present sCT-STPP ionic complex formulation thought to be a novel oral delivery candidate for the treatment of osteoporosis.

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

    DTIC Science & Technology

    1986-01-01

    amphibian sympathetic ganglion to inhibit the M current (8). ATP may affect - . dorsal root terminals in the toad spinal cord (343), and function...Perfusion Twenty-five frogs (Rana pipiens and Rana temporaria) were •de individually sacrificed by decapitation and pithing the spinal cord . During...various nucleosides and nucleotides on the isolated toad spinal cord . Gen. Pharmacol. 9:239-247, 1978. 344. Phillis, J.W. and Wu, P.H. The role of

  1. Importance of adenosine triphosphate in phospholipase A2-induced rabbit renal proximal tubule cell injury.

    PubMed Central

    Nguyen, V D; Cieslinski, D A; Humes, H D

    1988-01-01

    The pathogenesis of ischemic renal tubular cell injury involves a complex interaction of different processes, including membrane phospholipid alterations and depletion of high-energy phosphate stores. To assess the role of membrane phospholipid changes due to activation of phospholipases in renal tubule cell injury, suspensions enriched in rabbit renal proximal tubule segments were incubated with exogenous phospholipase A2 (PLA2). Exogenous PLA2 did not produce any significant change in various metabolic parameters reflective of cell injury in control nonhypoxic preparations despite a significant decrease in phosphatidylethanolamine (PE) and moderate increases in lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, exogenous PLA2 treatment of hypoxic tubules resulted in a severe degree of cell injury, as demonstrated by marked declines in tubule K+ and ATP contents and significant decreases in tubule uncoupled respiratory rates, and was associated with significant phospholipid alterations, including marked declines in phosphatidylcholine (PC) and PE and significant rises in LPC, LPE, and free fatty acids (FFA). The injurious metabolic effects of exogenous PLA2 on hypoxic tubules were reversed by addition of ATP-MgCl2 to the tubules. The protective effect of ATP-MgCl2 was associated with increases in tubule PC and PE contents and declines in LPC, LPE, and FFA contents. These experiments thus indicate that an increase in exogenous PLA2 activity produces renal proximal tubule cell injury when cell ATP levels decline, at which point phospholipid resynthesis cannot keep pace with phospholipid degradation with resulting depletion of phospholipids and accumulation of lipid by-products. High-energy phosphate store depletion appears to be an important condition for exogenous PLA2 activity to induce renal tubule cell injury. PMID:3417866

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

    PubMed

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

    2013-08-01

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

  3. Purinergic receptor activation inhibits osmotic glial cell swelling in the diabetic rat retina.

    PubMed

    Wurm, Antje; Iandiev, Ianors; Hollborn, Margrit; Wiedemann, Peter; Reichenbach, Andreas; Zimmermann, Herbert; Bringmann, Andreas; Pannicke, Thomas

    2008-10-01

    The anti-inflammatory glucocorticoid, triamcinolone acetonide, is used clinically for the rapid resolution of diabetic macular edema. Osmotic swelling of glial cells may contribute to the development of retinal edema. Triamcinolone inhibits the swelling of retinal glial cells of diabetic rats. Here, we determined whether the effect of triamcinolone is mediated by a receptor-dependent mechanism. Hyperglycemia was induced in rats with streptozotocin injection. After 6-10 months, the swelling properties of glial cells in retinal slices upon hypotonic challenge were determined. Nucleotide-degrading ecto-enzymes were immunostained in retinal slices and glial cells. Hypotonic challenge did not change the size of glial cell bodies from control retinas but induced swelling of cells from diabetic animals. Triamcinolone inhibited glial cell swelling; this effect was prevented by a selective antagonist of adenosine A1 receptors, an inhibitor of nucleoside transporters, inhibitors of adenylyl cyclase and protein kinase A activation, and inhibitors of potassium and chloride channels. In diabetic (but not control) retinas, the effect of triamcinolone apparently involves extracellular nucleotide degradation. Glial cells from diabetic retinas displayed immunolabeling against nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) which was not observed in control retinas. The mRNA expression for NTPDase1 was significantly increased in the retina of diabetic rats. It is suggested that triamcinolone induces the release and formation of endogenous adenosine that subsequently activates A1 receptors resulting in ion efflux through potassium and chloride channels and prevention of osmotic swelling. Whereas adenosine is liberated via facilitated transport in control retinas, an extracellular formation of adenosine contributes to the effect of triamcinolone in diabetic retinas.

  4. Release of ATP induced by hypertonic solutions in Xenopus oocytes

    PubMed Central

    Aleu, Jordi; Martín-Satué, Mireia; Navarro, Piedad; de Lara, Ivanna Pérez; Bahima, Laia; Marsal, Jordi; Solsona, Carles

    2003-01-01

    ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mm). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water. PMID:12562935

  5. Mechanical stimulation evokes rapid increases in extracellular adenosine concentration in the prefrontal cortex.

    PubMed

    Ross, Ashley E; Nguyen, Michael D; Privman, Eve; Venton, B Jill

    2014-07-01

    Mechanical perturbations can release ATP, which is broken down to adenosine. In this work, we used carbon-fiber microelectrodes and fast-scan cyclic voltammetry to measure mechanically stimulated adenosine in the brain by lowering the electrode 50 μm. Mechanical stimulation evoked adenosine in vivo (average: 3.3 ± 0.6 μM) and in brain slices (average: 0.8 ± 0.1 μM) in the prefrontal cortex. The release was transient, lasting 18 ± 2 s. Lowering a 15-μm-diameter glass pipette near the carbon-fiber microelectrode produced similar results as lowering the actual microelectrode. However, applying a small puff of artificial cerebral spinal fluid was not sufficient to evoke adenosine. Multiple stimulations within a 50-μm region of a slice did not significantly change over time or damage cells. Chelating calcium with EDTA or blocking sodium channels with tetrodotoxin significantly decreased mechanically evoked adenosine, signifying that the release is activity dependent. An alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, did not affect mechanically stimulated adenosine; however, the nucleoside triphosphate diphosphohydrolase 1,2 and 3 (NTDPase) inhibitor POM-1 significantly reduced adenosine so a portion of adenosine is dependent on extracellular ATP metabolism. Thus, mechanical perturbations from inserting a probe in the brain cause rapid, transient adenosine signaling which might be neuroprotective. We have discovered immediate changes in adenosine concentration in the prefrontal cortex following mechanical stimulation. The adenosine increase lasts only about 20 s. Mechanically stimulated adenosine was activity dependent and mostly because of extracellular ATP metabolism. This rapid, transient increase in adenosine may help protect tissue and would occur during implantation of any electrode, such as during deep brain stimulation.

  6. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

    PubMed

    Maloney, James P; Branchford, Brian R; Brodsky, Gary L; Cosmic, Maxwell S; Calabrese, David W; Aquilante, Christina L; Maloney, Kelly W; Gonzalez, Joseph R; Zhang, Weiming; Moreau, Kerrie L; Wiggins, Kerri L; Smith, Nicholas L; Broeckel, Ulrich; Di Paola, Jorge

    2017-03-16

    Ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) degrades the purines ATP and ADP that are key regulators of inflammation and clotting. We hypothesized that NTPDase1 polymorphisms exist and that they regulate this pathway. We sequenced the ENTPD1 gene (encoding NTPDase1) in 216 subjects then assessed genotypes in 2 cohorts comprising 2213 humans to identify ENTPD1 polymorphisms associated with venous thromboembolism (VTE). The G allele of the intron 1 polymorphism rs3176891 was more common in VTE vs. controls (odds ratio 1.26-1.9); it did not affect RNA splicing, but it was in strong linkage disequilibrium with the G allele of the promoter polymorphism rs3814159, which increased transcriptional activity by 8-fold. Oligonucleotides containing the G allele of this promoter region bound nuclear extracts more avidly. Carriers of rs3176891 G had endothelial cells with increased NTPDase1 activity and protein expression, and had platelets with enhanced aggregation. Thus, the G allele of rs3176891 marks a haplotype associated with increased clotting and platelet aggregation attributable to a promoter variant associated with increased transcription, expression, and activity of NTPDase1. We term this gain-of-function phenotype observed with rs3814159 G "CD39 Denver."-Maloney, J. P., Branchford, B. R., Brodsky, G. L., Cosmic, M. S., Calabrese, D. W., Aquilante, C. L., Maloney, K. W., Gonzalez, J. R., Zhang, W., Moreau, K. L., Wiggins, K. L., Smith, N. L., Broeckel, U., Di Paola, J. The ENTPD1 promoter polymorphism -860 A > G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk.

  7. Role for Apyrases in Polar Auxin Transport in Arabidopsis1[C][W][OA

    PubMed Central

    Liu, Xing; Wu, Jian; Clark, Greg; Lundy, Stacey; Lim, Minhui; Arnold, David; Chan, Jing; Tang, Wenqiang; Muday, Gloria K.; Gardner, Gary; Roux, Stanley J.

    2012-01-01

    Recent evidence indicates that extracellular nucleotides regulate plant growth. Exogenous ATP has been shown to block auxin transport and gravitropic growth in primary roots of Arabidopsis (Arabidopsis thaliana). Cells limit the concentration of extracellular ATP in part through the activity of ectoapyrases (ectonucleoside triphosphate diphosphohydrolases), and two nearly identical Arabidopsis apyrases, APY1 and APY2, appear to share this function. These findings, plus the fact that suppression of APY1 and APY2 blocks growth in Arabidopsis, suggested that the expression of these apyrases could influence auxin transport. This report tests that hypothesis. The polar movement of [3H]indole-3-acetic acid in both hypocotyl sections and primary roots of Arabidopsis seedlings was measured. In both tissues, polar auxin transport was significantly reduced in apy2 null mutants when they were induced by estradiol to suppress the expression of APY1 by RNA interference. In the hypocotyl assays, the basal halves of APY-suppressed hypocotyls contained considerably lower free indole-3-acetic acid levels when compared with wild-type plants, and disrupted auxin transport in the APY-suppressed roots was reflected by their significant morphological abnormalities. When a green fluorescent protein fluorescence signal encoded by a DR5:green fluorescent protein construct was measured in primary roots whose apyrase expression was suppressed either genetically or chemically, the roots showed no signal asymmetry following gravistimulation, and both their growth and gravitropic curvature were inhibited. Chemicals that suppress apyrase activity also inhibit gravitropic curvature and, to a lesser extent, growth. Taken together, these results indicate that a critical step connecting apyrase suppression to growth suppression is the inhibition of polar auxin transport. PMID:23071251

  8. Generation and Characterization of Specific Antibodies to the Murine and Human Ectonucleotidase NTPDase8

    PubMed Central

    Pelletier, Julie; Salem, Mabrouka; Lecka, Joanna; Fausther, Michel; Bigonnesse, François; Sévigny, Jean

    2017-01-01

    The ectonucleotidase nucleoside triphosphate diphosphohydrolase-8 (NTPDase8) is the last member of the Ecto-NTPDase family to be discovered and characterized. It is a transmembrane protein which regulates the concentration of the agonists of P1 and P2 receptors at the cell surface. The functions of the enzyme are still not known partly due to the lack of specific tools such as antibodies. In this work, guinea pig polyclonal antibodies against mouse NTPDase8 and mouse monoclonal antibodies against human NTPDase8 have been generated and characterized. For the production of antibodies against mouse NTPDase8 several techniques have been tried. Several peptide antigens in several hosts (rabbit, rat, hamster, and guinea pig) failed to give a positive reaction suggesting that NTPDase8 is poorly immunogenic. In this study, we describe the successful process that led to anti-mouse NTPDase8, namely the cDNA immunization technique. Monoclonal antibodies to human NTPDase8 were also obtained by cDNA immunization followed by a final injection with transfected human embryonic kidney (HEK 293T) cells expressing human NTPDase8. The specificity of these antibodies was evaluated by Western blot, immunocytochemistry, immunohistochemistry and flow cytometry. In contrast, all commercial antibodies to NTPDase8 peptides that we have tested failed to give a specific positive signal against the expressed NTPDase8 protein when used to probe Western blots. In addition, immunohistochemistry experiments confirmed the presence of NTPDase8 in mouse liver canaliculi. The tools generated in this work will help characterize NTPDase8 localization and function in future studies and its contribution to the modulation of P1 and P2 receptor activation. PMID:28337144

  9. Highly Potent and Selective Ectonucleotide Pyrophosphatase/Phosphodiesterase I Inhibitors Based on an Adenosine 5′-(α or γ)- Thio-(α,β- or β,γ)-methylenetriphosphate Scaffold

    PubMed Central

    Nadel, Yael; Lecka, Joanna; Gilad, Yocheved; Ben-David, Gal; Förster, Daniel; Reiser, Georg; Kenigsberg, Sarah; Camden, Jean; Weisman, Gary A.; Senderowitz, Hanoch; Sévigny, Jean; Fischer, Bilha

    2015-01-01

    Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure–activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ- CH2 (1), ATP-α-thio-β,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1–3 at 100 μM inhibited thymidine 5′-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23–43%, respectively, and only slightly affected (0–40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 μM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 μM. Analogues 1–3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1–3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors. PMID:24846781

  10. Gene Expression and Activity Profiling Reveal a Significant Contribution of Exo-Phosphotransferases to the Extracellular Nucleotides Metabolism in HUVEC Endothelial Cells.

    PubMed

    Wujak, Magdalena; Hetmann, Anna; Porowińska, Dorota; Roszek, Katarzyna

    2016-11-11

    Purinergic signaling maintains local tissue homeostasis in blood vessels via the regulation of vascular tone, blood platelet aggregation, cell proliferation, and differentiation as well as inflammatory responses. Extracellular purines are important signaling molecules in the vasculature, and both purine-hydrolysing as well as -phosphorylating enzymes are considered to selectively govern extracellular nucleotide/nucleoside metabolism. Recent studies have provided some evidence for the existence of these enzymes in a soluble form in human blood and their secretion into the extracellular space under physiological and pathological conditions. However, the comprehensive analysis of endothelium-derived enzymes involved in purine metabolic pathways has received no attention so far. In the presented study, in vitro cultured human umbilical vein endothelial cells (HUVEC) are shown to be an abundant source of exo-nucleotidases comprising 5'-nucleotidase (exo-5'-NT), and nucleoside triphosphate diphosphohydrolases (exo-NTPDase) as well as phosphotransferases, represented by nucleoside diphosphate kinase (exo-NDPK) and adenylate kinase (exo-AK). An attempt is also made to demonstrate that, in contrast to the metabolic pattern determined on the endothelial cell surface, exo-phosphorylating activities markedly predominate over exo-hydrolytic ones. We present for the first time the expression profiles of genes encoding isoenzymes belonging to distinct nucleotide kinase and nucleotidase families. The genes encoding NDPK1, NDPK2, AK1, and AK2 phosphotransferases have been shown to be expressed at the highest level in HUVEC cells. The data indicate the coexistence of secreted and cell-associated factors of endothelial origin mediating ATP-consuming and ATP-generating pathways with the predominance of exo-phosphotransferases activity. The described enzymes contribute to the regulation of purinergic signal duration and extent in the venous vasculature. J. Cell. Biochem. 9999: 1

  11. Ectonucleotidases as regulators of purinergic signaling in thrombosis, inflammation, and immunity.

    PubMed

    Deaglio, Silvia; Robson, Simon C

    2011-01-01

    Evolving studies in models of transplant rejection, inflammatory bowel disease, and cancer, among others, have implicated purinergic signaling in clinical manifestations of vascular injury and thrombophilia, inflammation, and immune disturbance. Within the vasculature, spatial and temporal expression of CD39 nucleoside triphosphate diphosphohydrolase (NTPDase) family members together with CD73 ecto-5'-nucleotidase control platelet activation, thrombus size, and stability. This is achieved by closely regulated phosphohydrolytic activities to scavenge extracellular nucleotides, maintain P2-receptor integrity, and coordinate adenosinergic signaling responses. The CD38/CD157 family of extracellular NADases degrades NAD(+) and generates Ca(2+)-active metabolites, including cyclic ADP ribose and ADP ribose. These mediators regulate leukocyte adhesion and chemotaxis. These mechanisms are crucial in vascular homeostasis, hemostasis, thrombogenesis, and during inflammation. There has been recent interest in ectonucleotidase expression by immune cells. CD39 expression identifies Langerhans-type dendritic cells and efficiently distinguishes T regulatory cells from other resting or activated T cells. CD39, together with CD73 in mice, serves as an integral component of the suppressive machinery of T cells. Purinergic responses also impact generation of T helper-type 17 cells. Further, CD38 and changes in NAD(+) availability modulate ADP ribosylation of the cytolytic P2X7 receptor that deletes T regulatory cells. Expression of CD39, CD73, and CD38 ectonucleotidases on either endothelial or immune cells allows for homeostatic integration and control of vascular inflammatory and immune cell reactions at sites of injury. Ongoing development of therapeutic strategies targeting these and other ectonucleotidases offers promise for the management of vascular thrombosis, disordered inflammation, and aberrant immune reactivity.

  12. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  13. Effects of 8-halo-7-deaza-2'-deoxyguanosine triphosphate on DNA synthesis by DNA polymerases and cell proliferation.

    PubMed

    Yin, Yizhen; Sasaki, Shigeki; Taniguchi, Yosuke

    2016-08-15

    8-OxodG (8-oxo-2'-deoxyguanosine) is representative of nucleoside damage and shows a genotoxicity. To significantly reveal the contributions of 7-NH and C8-oxygen to the mutagenic effect of 8-oxodG by DNA polymerases, we evaluated the effects of the 8-halo-7-deaza-dG (8-halogenated 7-deaza-2'-deoxyguanosine) derivatives by DNA polymerases. 8-Halo-7-deaza-dGTPs were poorly incorporated by both KF(exo(-)) and human DNA polymerase β opposite dC or dA into the template DNA. Furthermore, it was found that KF(exo(-)) was very sensitive to the introduction of the C8-halogen, while polymerase β can accommodate the C8-halogen resulting in an efficient dCTP insertion opposite the 8-halo-7-deaza-dG in the template DNA. These results indicate that strong hydrogen bonding between 7-NH in the 8-oxo-G nucleobase and 1-N in the adenine at the active site of the DNA polymerase is required for the mutagenic effects. Whereas, I-deaza-dGTP shows an antiproliferative effect for the HeLa cells, suggesting that it could become a candidate as a new antitumor agent.

  14. Nuclear Overhauser effect studies on the conformation of magnesium adenosine 5'-triphosphate bound to rabbit muscle creatine kinase

    SciTech Connect

    Rosevear, P.R.; Powers, V.M.; Dowhan, D.; Mildvan, A.S.; Kenyon, G.L.

    1987-08-25

    Nuclear Overhauser effects were used to determine interproton distances on MgATP bound to rabbit muscle creatine kinase. The internuclear distances were used in a distance geometry program that objectively determines both the conformation of the bound MgATP and its uniqueness. Two classes of structures were found that satisfied the measured interproton distances. Both classes had the same anti glycosidic torsional angle (X = 78 +/- 10/sup 0/) but differed in their ribose ring puckers (O1'-endo or C4'-exo). The uniqueness of the glycosidic torsional angle is consistent with the preference of creatine kinase for adenine nucleotides. One of these conformations of MgATP bound to creatine kinase is indistinguishable from the conformation found for Co(NH/sub 3/)/sub 4/ ATP bound to the catalytic subunit of protein kinase, which also has a high specificity for adenine nucleotides. Distance geometry calculations also suggest that upper limit distances, when low enough (less than or equal to 3.4 A), can be used instead of measured distances to define, within experimental error, the glycosidic torsional angle of bound nucleotides. However, this approach does not permit an evaluation of the ribose ring pucker.

  15. An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range.

    PubMed

    Wei, Yanli; Chen, Yanxia; Li, Huanhuan; Shuang, Shaomin; Dong, Chuan; Wang, Gufeng

    2015-01-15

    A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.

  16. Rapid and direct detection of attomole adenosine triphosphate (ATP) by MALDI-MS using rutile titania chips.

    PubMed

    Manikandan, Muthu; Hasan, Nazim; Wu, Hui-Fen

    2012-11-07

    We report the rutile titania-based capture of ATP and its application as a MALDI-MS target plate. This chip, when immersed in solutions containing different concentrations of ATP, can capture ATP and lead to its successful detection in MALDI-MS. We have optimized the ideal surface, showing an increased capture efficacy of the 900 °C (rutile) titania surfaces. We demonstrate the use of this chip as a target plate for direct analysis of the attached ATP using MALDI-MS, down to attomolar concentrations. This chip has a promising future for the detection of ATP in environmental samples, which may eventually be used as a pollution indicator in particular environments.

  17. [Effect of prolonged propofol infusion on myocardial enzyme, mitochondrial cytochrome C and adenosine 
triphosphate in rabbits].

    PubMed

    Xu, Guangmin; Lan, Zhixun; Tong, Xianxiang

    2016-11-28

    目的:探讨长时间输注丙泊酚对兔心肌酶、线粒体细胞色素C和ATP的影响。方法:18只成年新西兰兔随机分为空白对照组、丙泊酚组和脂肪乳组,每组6只。空白对照组持续输注0.9%生理盐水,丙泊酚组持续输注1%丙泊酚,脂肪乳组持续输注10%脂肪乳。试验过程中分别在0,8,16 h和实验终点采集动脉血检测肌酸激酶(creatine kinase,CK)和肌酸激酶同工酶(creatine kinase-MB,CK-MB),实验结束取心肌组织,用差速离心法分离心肌线粒体,高效液相色谱法测量线粒体细胞色素C含量和ATP含量。结果:与空白对照组相比,丙泊酚组和脂肪乳组细胞色素C从线粒体的释放增加,差异均有统计学意义(均P<0.05);丙泊酚组和脂肪组两组间比较差异无统计学意义(P>0.05);3组间线粒体内ATP含量比较,差异均无统计学意义(均P>0.05)。与同组0 h点比较,丙泊酚组和脂肪乳组在输注8,16和24 h的CK升高(均P<0.05);与空白对照组比较,丙泊酚组和脂肪乳组在输注8,16和
24 h的CK升高(均P<0.05);与脂肪乳组比较,丙泊酚组CK在输注8,16和24 h升高,差异均有统计学意义(均P<0.05)。与空白对照组比较,丙泊酚组在持续输注丙泊酚24 h时CK-MB升高明显(P<0.05);与同组0 h点比较,丙泊酚组在持续输注24 h后CK-MB升高明显(P<0.05)。结论:长时间输注丙泊酚和脂肪乳均可使血清CK升高,但丙泊酚可使CK-MB升高。丙泊酚和脂肪乳均能使心肌线粒体细胞色素C释放增加,但不影响心肌线粒体ATP产量。.

  18. Adenosine triphosphate (ATP) levels in paracetamol-induced cell injury in the rat in vivo and in vitro.

    PubMed

    Martin, F L; McLean, A E

    1995-12-15

    We have investigated the relationship between ATP levels and the onset and progression of cell injury induced by paracetamol overdose both in vivo and in vitro. Liver slices obtained from phenobarbitone-induced and non-induced rats were used in a model in vitro system. Slices were exposed to paracetamol (2-10 mM), for 120 min and then incubated without paracetamol for a further 240 min. ATP levels are reduced upon exposure to paracetamol in liver slices from both phenobarbitone-induced and non-induced rats. Cell injury, as quantified by measuring leakage of lactate dehydrogenase (LDH) and potassium (K+), does not become apparent until 240 min, some 120 min after exposure to paracetamol had ended. This irreversible cell injury is not observed in liver slices from non-induced rats. For in vivo studies rats were phenobarbitone-induced and received i.p. injections of 800 mg/kg body weight paracetamol. Hepatic ATP levels were measured and are found to drop sharply by 3 h post-injection. Development of irreversible hepatic cell injury was assessed by measuring serum enzyme (ALT) activity. ALT levels do not rise until 12 h have elapsed. Paracetamol in overdose gives rise to ATP depletion in liver cells, that is early, independent of paracetamol metabolism and probably spread throughout the lobule. In contrast cell injury is found late and only in our phenobarbitone-induced rats. No cell injury is observed in liver slices from non-induced rats. This suggests that while the level of ATP depletion which is observed may be a necessary part of cell injury by paracetamol, it is not a sufficient cause.

  19. Properties of flagellar "rigor waves" formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm.

    PubMed

    Gibbons, B H; Gibbons, I R

    1974-12-01

    Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 microM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 microM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.

  20. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    PubMed Central

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.

    2016-01-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

  1. Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver.

    PubMed Central

    Moreno, F J; Sánchez-Urrutia, L; Medina, J M; Sánchez-Medina, F; Mayor, F

    1975-01-01

    1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably less than the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP. PMID:173301

  2. Physicochemical properties and supernucleophilicity of oxime-functionalized surfactants: hydrolytic catalysts toward dephosphorylation of di- and triphosphate esters.

    PubMed

    Singh, Namrata; Karpichev, Yevgen; Gupta, Bhanushree; Satnami, Manmohan L; Marek, Jan; Kuca, Kamil; Ghosh, Kallol K

    2013-04-11

    Aggregation and kinetic studies have been performed to understand the hydrolytic potencies of the series of oxime-functionalized surfactants, viz., 3- hydroxyiminomethyl-1-alkylpyridinium bromide (alkyl = CnH2n+1, n = 10, 12, 14, 16, 18) in the cleavage of phosphate esters, p-nitrophenyl diphenyl phosphate (PNPDPP) and bis(2,4-dinitrophenyl) phosphate (BNDPP), in mixed micelles with cetylpyridinium bromide (CPB). Micellization and surface properties of mixed micelles functional surfactants with CPB were studied by conductivity and surface tension measurements. Acid dissociation constants (pKa) were determined, the effect of functional surfactant alkyl chain length and pH on the observed rate constant (kobs) for phosphate ester cleavage has been discussed, and the effect of substrate on the supernucleophilicities of the studied oximes was monitored. Functionalized oxime-based surfactants were proved to be supernucleophiles to attack on the P═O center of tri- and diphosphate esters. Oximes with hexadecyl alkyl chain length (3-C16) showed maximum micellar effect on the rate constants toward PNPDPP. Micellar effects were analyzed in terms of the pseudophase model.

  3. Inotropic and coronary vasodilatory actions of the K-adenosine triphosphate channel modulator nicorandil in human tissue.

    PubMed

    Müller-Ehmsen, J; Brixius, K; Hoischen, S; Schwinger, R H

    1996-12-01

    The present study aimed to characterize the inotropic and vasodilatory properties of the K-ATP channel opener nicorandil (NIC) in isolated human cardiac tissue. For comparison, the Ca+2 channel blockers diltiazem (DIL) and nifedipine (NIF) have been studied. Concentration-dependent effects of NIC, DIL and NIF on the force of contraction (FOC) and the vascular tone have been studied on left ventricular papillary muscle strips (dilated cardiomyopathy, New York Heart Association Class IV, n = 20; nonfailing, donor hearts, n = 4), on right auricular trabeculae (nonfailing, n = 5) and on precontracted (prostaglandin F2 alpha: 0.3, 0.5 or 1 mumol/l) isolated human coronary artery rings (cardiac transplantation, n = 15). NIC, DIL and NIF concentration-dependently reduced the FOC of the papillary muscle preparations. However, the IC25 for the negative inotropic effect was significantly higher for NIC compared to DIL and NIF. The maximal negative inotropic effects of NIC, DIL and NIF (100 mumol/l) were -48.2 +/- 4.1, -92.9 +/- 0.9 and -93.4 +/- 1.4% of the basal FOC. The negative inotropic actions of NIC were similar in the human failing and the nonfailing ventricular and in the right atrial myocardium. Whereas pretreatment with methylene blue, an inhibitor of guanylyl cyclase, had no effect on the negative inotropic action of NIC, it was almost abolished by glibenclamide, a selective antagonist of the ATP-dependent K channels. NIC, DIL and NIF relaxed the coronary artery rings with 97.1 +/- 0.5, 90.7 +/- 0.9 and 96.4 +/- 0.7% of maximal relaxation (papaverine, 100 mumol/l). The rank order of vasodilatory potency was NIF > NIC > DIL. In conclusion, NIC is as effective as DIL and NIF in relaxing human coronary artery rings. However, NIC showed significantly lower negative inotropic effects when compared with the Ca+2 channel antagonists. The negative inotropic action of NIC is probably due to an interaction with the ATP-dependent K channels. In addition, activation of guanylyl cyclase does not seem to exert any negative inotropic action in the human myocardium.

  4. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

    PubMed Central

    1995-01-01

    In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo. PMID:7490281

  5. The Relation of the 515 Nanometers Absorbance Change to Adenosine Triphosphate Formation in Chloroplasts and Digitonin Subchloroplast Particles 1

    PubMed Central

    Neumann, Joseph; Ke, Bacon; Dilley, Richard A.

    1970-01-01

    The flash-induced absorbance changes at 515 nanometers has been studied in chloroplasts and in digitonin subchloroplast particles of lettuce. The effect of various conditions and uncouplers was tested on the decay kinetics of this absorbance change and on ATP formation in the presence of phenazine methosulphate, either by continuous or flash illumination. It has been found that in chloroplasts, carbonyl cyanide m-chloromethoxyphenylhydrazone and nigericin in the presence of K+ accelerate the decay of the 515 change and inhibit ATP formation. However, under a variety of conditions the rate of decay of the 515 absorbance change was found to be unrelated to ATP formation. Preillumination, addition of valinomycin in the presence of K+, addition of Na+, or divalent cations accelerate the decay of the 515 absorbance change markedly but have no effect on ATP formation. Addition of phosphorylation reagents has no effect on the decay rate beyond that obtained by Mg2+ and inorganic phosphate. NH4Cl, and to some extent atebrin, while inhibiting ATP formation, do not affect the decay of the 515 absorbance change. In digitonin subchloroplast particles the decay kinetics of the absorbance change resemble that of chloroplasts, but the magnitude of the change is smaller. The pH change in this preparation is reduced much more than the 515 absorbance change. According to the chemiosmotic hypothesis, the sum of ΔE(membrane potential) and ΔpH is the driving force for ATP formation. The lack of an increase in ΔE in digitonin subchloroplast particles, which are practically devoid of ΔpH and have a normal ATP-forming activity, is inconsistent with the chemiosmotic hypothesis. PMID:16657427

  6. Adenosine 5'-triphosphate consumption by smooth muscle as predicted by the coupled four-state crossbridge model.

    PubMed Central

    Hai, C M; Murphy, R A

    1992-01-01

    We have proposed a four-state crossbridge model to explain contraction and the latch state in arterial smooth muscle. Ca(2+)-dependent crossbridge phosphorylation was the only postulated regulatory mechanism and the latchbridge (a dephosphorylated, attached crossbridge) was the only novel element in the model. In this study, we used the model to predict rates of ATP consumption by crossbridge phosphorylation (JPhos) and cycling (JCycle) during isometric and isotonic contractions in arterial smooth muscle; then we compared model predictions with experimental data. The model predicted that JPhos and JCycle were similar in magnitude in isometric contractions, and both increased almost linearly with myosin phosphorylation. The predicted relationship between isometric stress and ATP consumption was quasihyperbolic, but approximately linear when myosin phosphorylation was below 35%, in agreement with most of the available data. Muscle shortening increased the predicted values of JCycle up to 3.7-fold depending on shortening velocity and the level of myosin phosphorylation. The predicted maximum work output per ATP was 7.4-7.8 kJ/mol ATP and was relatively insensitive to changes in myosin phosphorylation. The predicted increase in JCycle with shortening was in agreement with available data, but the model prediction that work output per ATP was insensitive to changes in myosin phosphorylation was unexpected and remains to be tested in future experiments. PMID:1547336

  7. Inositol 1,4,5-triphosphate drives glutamatergic and cholinergic inhibition selectively in spiny projection neurons in the striatum.

    PubMed

    Clements, Michael A; Swapna, Immani; Morikawa, Hitoshi

    2013-02-06

    The striatum is critically involved in the selection of appropriate actions in a constantly changing environment. The spiking activity of striatal spiny projection neurons (SPNs), driven by extrinsic glutamatergic inputs, is shaped by local GABAergic and cholinergic networks. For example, it is well established that different types of GABAergic interneurons, activated by extrinsic glutamatergic and local cholinergic inputs, mediate powerful feedforward inhibition of SPN activity. In this study, using mouse striatal slices, we show that glutamatergic and cholinergic inputs exert direct inhibitory regulation of SPN activity via activation of metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors. While pressure ejection of the group I mGluR (mGluR1/5) agonist DHPG [(S)-3,5-dihydroxyphenylglycine] equally engages both mGluR1 and mGluR5 subtypes, the mGluR-dependent component of IPSCs elicited by intrastriatal electrical stimulation is almost exclusively mediated by the mGluR1 subtype. Ca(2+) release from intracellular stores specifically through inositol 1,4,5-triphospahte receptors (IP(3)Rs) and not ryanodine receptors (RyRs) mediates this form of inhibition by gating two types of Ca(2+)-activated K(+) channels (i.e., small-conductance SK channels and large-conductance BK channels). Conversely, spike-evoked Ca(2+) influx triggers Ca(2+) release solely through RyRs to generate SK-dependent slow afterhyperpolarizations, demonstrating functional segregation of IP(3)Rs and RyRs. Finally, IP(3)-induced Ca(2+) release is uniquely observed in SPNs and not in different types of interneurons in the striatum. These results demonstrate that IP(3)-mediated activation of SK and BK channels provides a robust mechanism for glutamatergic and cholinergic inputs to selectively suppress striatal output neuron activity.

  8. Inositol 1,4,5-Triphosphate Drives Glutamatergic and Cholinergic Inhibition Selectively in Spiny Projection Neurons in the Striatum

    PubMed Central

    Clements, Michael A.; Swapna, Immani; Morikawa, Hitoshi

    2013-01-01

    The striatum is critically involved in the selection of appropriate actions in a constantly changing environment. The spiking activity of striatal spiny projection neurons (SPNs), driven by extrinsic glutamatergic inputs, is shaped by local GABAergic and cholinergic networks. For example, it is well established that different types of GABAergic interneurons, activated by extrinsic glutamatergic and local cholinergic inputs, mediate powerful feedforward inhibition of SPN activity. In this study, using mouse striatal slices, we show that glutamatergic and cholinergic inputs exert direct inhibitory regulation of SPN activity via activation of metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors. While pressure ejection of the group I mGluR (mGluR1/5) agonist DHPG [(S)-3,5-dihydroxyphenylglycine] equally engages both mGluR1 and mGluR5 subtypes, the mGluR-dependent component of IPSCs elicited by intrastriatal electrical stimulation is almost exclusively mediated by the mGluR1 subtype. Ca2+ release from intracellular stores specifically through inositol 1,4,5-triphospahte receptors (IP3Rs) and not ryanodine receptors (RyRs) mediates this form of inhibition by gating two types of Ca2+ -activated K+ channels (i.e., small-conductance SK channels and large-conductance BK channels). Conversely, spike-evoked Ca2+ influx triggers Ca2+ release solely through RyRs to generate SK-dependent slow afterhyperpolarizations, demonstrating functional segregation of IP3Rs and RyRs. Finally, IP3-induced Ca2+ release is uniquely observed in SPNs and not in different types of interneurons in the striatum. These results demonstrate that IP3-mediated activation of SK and BK channels provides a robust mechanism for glutamatergic and cholinergic inputs to selectively suppress striatal output neuron activity. PMID:23392696

  9. Overexpression of CD39 in hepatocellular carcinoma is an independent indicator of poor outcome after radical resection

    PubMed Central

    Cai, Xiao-Yan; Ni, Xiao-Chun; Yi, Yong; He, Hong-Wei; Wang, Jia-Xing; Fu, Yi-Peng; Sun, Jian; Zhou, Jian; Cheng, Yun-Feng; Jin, Jian-Jun; Fan, Jia; Qiu, Shuang-Jian

    2016-01-01

    Abstract Nucleoside triphosphate diphosphohydrolase-1 (ENTPD1/CD39) is the rate-limiting enzyme in a cascade leading to the generation of immunosuppressive adenosine and plays an important role in tumor progression. This study aimed to evaluate the expression of CD39 and CD39+Foxp3+ regulatory T cells (Tregs) and to determine their prognostic role in patients with hepatocellular carcinoma (HCC) after radical resection. Immunohistochemistry (IHC) and double IHC were used to analyze CD39 expression or the expression of CD39 and Foxp3 in a cohort of 324 HCC patients who underwent curative resection. The quantification of CD39 expression levels was determined using a computerized image analysis system and was evaluated by mean optical density (MOD), which corresponded to the positive staining intensity of CD39. The number of positive Foxp3 cells and both CD39 and Foxp3 positive cells in each 1-mm-diameter cylinder were counted under high-power magnification (×400). The “minimum P value” approach was used to obtain the optimal cutoff value for the best separation between groups of patients in relation to time to recurrence (TTR) or overall survival (OS). The expression of CD39 in HCC cell lines with stepwise metastatic potential and in human umbilical vein endothelial cells was determined by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence. The SPSS 17.0 statistical package was used for statistics. CD39 was principally expressed on vascular endothelial cells, macrophagocytes, Tregs, and tumor cells in HCC. Compared with paired peritumoral tissues, tumoral tissues had a significantly higher expression level of CD39 (P < 0.0001). Overexpression of tumoral CD39 was related to increased tumor recurrence and shortened overall survival. Furthermore, the expression level of peritumoral CD39 showed a prognostic role in TTR and OS. Double IHC showed that tumoral tissues had significantly higher Foxp3+Tregs and CD39+Foxp3+Tregs

  10. Genetic analysis of an allergic rhinitis cohort reveals an intercellular epistasis between FAM134B and CD39

    PubMed Central

    2014-01-01

    Background Extracellular ATP is a pro-inflammatory molecule released by damaged cells. Regulatory T cells (Treg) can suppress inflammation by hydrolysing this molecule via ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), also termed as CD39. Multiple studies have reported differences in CD39+ Treg percentages in diseases such as multiple sclerosis, Hepatitis B and HIV-1. In addition, CD39 polymorphisms have been implicated in immune-phenotypes such as susceptibility to inflammatory bowel disease and AIDS progression. However none of the studies published so far has linked disease-associated variants with differences in CD39 Treg surface expression. This study aims at identifying variants affecting CD39 expression on Treg and at evaluating their association with allergic rhinitis, a disease characterized by a strong Treg involvement. Methods Cohorts consisting of individuals of different ethnicities were employed to identify any association of CD39 variants to surface expression. Significant variant(s) were tested for disease association in a published GWAS cohort by one-locus and two-locus genetic analyses based on logistic models. Further functional characterization was performed using existing microarray data and quantitative RT-PCR on sorted cells. Results Our study shows that rs7071836, a promoter SNP in the CD39 gene region, affects the cell surface expression on Treg cells but not on other CD39+ leukocyte subsets. Epistasis analysis revealed that, in conjunction with a SNP upstream of the FAM134B gene (rs257174), it increased the risk of allergic rhinitis (P = 1.98 × 10-6). As a promoter SNP, rs257174 controlled the expression of the gene in monocytes but, notably, not in Treg cells. Whole blood transcriptome data of three large cohorts indicated an inverse relation in the expression of the two proteins. While this observation was in line with the epistasis data, it also implied that a functional link must exist. Exposure of monocytes to

  11. Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study

    PubMed Central

    2014-01-01

    Background Cordyceps cicadae is a medicinal fungus that is often used for treating cancer. However, the anticancer mechanisms of C. cicadae are largely unknown. This study aims to investigate the anticancer mechanisms of C. cicadae against hepatocellular carcinoma cells in vitro using a proteomic approach. Methods Human hepatocellular carcinoma MHCC97H cells were treated with a water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h and harvested for cell viability assays. The significant differences in protein expression between control and C. cicadae-treated cells were analyzed by two-dimensional gel-based proteomics coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Flow cytometry analysis was employed to investigate the cell cycle and cell death. The anticancer molecular mechanism was analyzed by whole proteome mapping. Results The water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) inhibited the growth of MHCC97H cells in a dose-dependent manner via G2/M phase cell cycle arrest with no evidence of apoptosis. Among the identified proteins with upregulated expression were dynactin subunit 2, N-myc downstream-regulated gene 1, heat shock protein beta-1, alpha-enolase isoform 1, phosphatidylinositol transfer protein, and WD repeat-containing protein 1. Meanwhile, the proteins with downregulated expression were 14-3-3 gamma, BUB3, microtubule-associated protein RP/EB family member 1, thioredoxin-like protein, chloride intracellular channel protein 1, ectonucleoside triphosphate diphosphohydrolase 5, xaa-Pro dipeptidase, enoyl-CoA delta isomerase 1, protein-disulfide isomerase-related chaperone Erp29, hnRNP 2H9B, peroxiredoxin 1, WD-40 repeat protein, and serine/threonine kinase receptor-associated protein. Conclusion The water extract of C. cicadae reduced the growth of human hepatocellular carcinoma MHCC97H cells via G2/M cell cycle arrest. PMID:24872842

  12. Pasteurella multocida toxin activates the inositol triphosphate signaling pathway in Xenopus oocytes via G(q)alpha-coupled phospholipase C-beta1.

    PubMed

    Wilson, B A; Zhu, X; Ho, M; Lu, L

    1997-01-10

    Pasteurella multocida toxin (PMT) has been hypothesized to cause activation of a GTP-binding protein (G-protein)-coupled phosphatidylinositol-specific phospholipase C (PLC) in intact cells. We used voltage-clamped Xenopus oocytes to test for direct PMT-mediated stimulation of PLC by monitoring the endogenous Ca2+-dependent C1- current. Injection of PMT induced an inward, two-component Cl- current, similar to that evoked by injection of IP3 through intracellular Ca2+ mobilization and Ca2+ influx through voltage-gated Ca2+ channels. These PMT-induced currents were blocked by specific inhibitors of Ca2+ and Cl- channels, removal of extracellular Ca2+, or chelation of intracellular Ca2+. Specific antibodies directed against an N-terminal, but not a C-terminal, peptide of PMT inhibited the toxin-induced currents, implicating that the N terminus of PMT is important for toxin activity. Injection with specific antibodies against PLCbeta1, PLCbeta2, PLCbeta3, or PLCgamma1 identified PLCbeta1 as the primary mediator of the PMT-induced Cl- currents. Injection with guanosine 5'-O-(2-(thio)diphosphate), antibodies to the common GTP-binding region of G-protein alpha subunits, or antibodies to different regions of G-protein beta subunits established the involvement of a G-protein alpha subunit in PMT-activation of PLCbeta1. Injection with specific antibodies against the alpha-subunits of G(q/11), G(s/olf), G(i/o/t/z), or G(i-1/i-2/i-3) isoforms confirmed the involvement of Gq/11alpha. Preinjection of oocytes with pertussis toxin enhanced the PMT response. Overexpression of G(q)alpha in oocytes could enhance the PMT response by 30-fold to more than 300-fold, whereas introduction of antisense G(q)alpha cRNA reduced the response by 7-fold. The effects of various specific antibodies on the PMT response were reproduced in oocytes overexpressing G(q)alpha.

  13. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    PubMed

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  14. Studies on yeast nucleoside triphosphate-nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). IV. Steady-state kinetic properties with thymidine nucleotides (including 3'-azido-3'-deoxythymidine analogues).

    PubMed

    Kuby, S A; Fleming, G; Alber, T; Richardson, D; Takenaka, H; Hamada, M

    1991-01-01

    A study of the steady-state kinetics of the crystalline brewer's yeast (Saccharomyces carlsbergensis) nucleoside diphosphokinase, with the magnesium complexes of the adenine and thymidine nucleotides as reactants, has led to a postulated kinetic mechanism which proceeds through a substituted enzyme. This agrees with the earlier conclusions of Garces and Cleland [Biochemistry 1969; 8:633-640] who characterized a reaction between the magnesium complexes of the adenine and uridine nucleotides. An advantage of using thymidine nucleotides as reactants is that they permit accurate, rapid and continuous assays of the enzymatic activity in coupled-enzymatic tests. Through measurements of the initial velocities and product inhibition studies, the Michaelis constants, maximum velocities, and inhibition constants could be evaluated for the individual substrates. Competitive substrate inhibition was encountered at relatively high substrate concentrations, which also permitted an evaluation of their ability to act as 'dead-end' inhibitors. The Michaelis constants for the 3'-azido-3'-deoxythymidine (AzT) analogues were also evaluated and, although these values were only somewhat higher than those of their natural substrates, the Km's for the adenine nucleotides as paired substrates were lower and the Vmax's were drastically reduced. The pharmacological implications of these observations are touched upon and extrapolated to the cases where therapeutic doses of AzT may be employed.

  15. The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization.

    PubMed

    Maisel, Tobias; Joseph, Stephanie; Mielke, Thorsten; Bürger, Jörg; Schwarzinger, Stephan; Meyer, Ortwin

    2012-01-01

    CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO₂] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean K(M) value of 0.69±0.14 mM ATP and an apparent V(max) value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO₂] cluster assembly are discussed.

  16. Hyaluronan Upregulates Mitochondrial Biogenesis and Reduces Adenoside Triphosphate Production for Efficient Mitochondrial Function in Slow-Proliferating Human Mesenchymal Stem Cells.

    PubMed

    Solis, Mairim Alexandra; Wei, Yau-Huei; Chang, Chiung-Hsin; Yu, Chen-Hsiang; Kuo, Pao-Lin; Huang, Lynn L H

    2016-10-01

    Hyaluronan-coated surfaces preserve the proliferation and differentiation potential of mesenchymal stem cells by prolonging their G1-phase transit, which maintains cells in a slow-proliferative mode. Mitochondria are known to play a crucial role in stem cell self-renewal and differentiation. In this study, for the first time, the metabolic mechanism underlying the hyaluronan-regulated slow-proliferative maintenance of stem cells was investigated by evaluating mitochondrial functions. Human placenta-derived mesenchymal stem cells (PDMSCs) cultured on hyaluronan-coated surfaces at 0.5, 3.0, 5.0, and 30 µg/cm(2) were found to have an average 58% higher mitochondrial mass and an increase in mitochondrial DNA copy number compared to noncoated tissue culture surfaces (control), as well as a threefold increase in the gene expression of the mitochondrial biogenesis-related gene PGC-1α. Increase in mitochondrial biogenesis led to a hyaluronan dose-dependent increase in mitochondrial membrane potential, ATP content, and oxygen consumption rate, with reactive oxygen species levels shown to be at least three times lower compared to the control. Although hyaluronan seemed to favor mitochondrial function, cell entry into a hyaluronan-regulated slow-proliferative mode led to a fivefold reduction in ATP production and coupling efficiency levels. Together, these results suggest that hyaluronan-coated surfaces influence the metabolic proliferative state of stem cells by upregulating mitochondrial biogenesis and function with controlled ATP production. This more efficiently meets the energy requirements of slow-proliferating PDMSCs. A clear understanding of the metabolic mechanism induced by hyaluronan in stem cells will allow future applications that may overcome the current limitations faced in stem cell culture. Stem Cells 2016;34:2512-2524.

  17. Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

    PubMed

    Jacobson, K B; Manos, R E

    1989-05-15

    The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.

  18. Detection of adenosine triphosphate in HeLa cell using capillary electrophoresis-laser induced fluorescence detection based on aptamer and graphene oxide.

    PubMed

    Fang, Bi-Yun; Yao, Ming-Hao; Wang, Chun-Yuan; Wang, Chao-Yang; Zhao, Yuan-Di; Chen, Fang

    2016-04-01

    A method for ATP quantification based on dye-labeled aptamer/graphene oxide (aptamer/GO) using capillary electrophoresis-laser induced fluorescence (CE-LIF) detecting technique has been established. In this method, the carboxyfluorescein (FAM)-labelled ATP aptamers were adsorbed onto the surface of GO, leading to the fluorescence quenching of FAM; after the incubation with a limited amount of ATP, stronger affinity between ATP aptamer and ATP resulted in the desorption of aptamers and the fluorescence restoration of FAM. Then, aptamer-ATP complex and excess of aptamer/GO and GO were separated and quantified by CE-LIF detection. It was shown that a linear relation was existing in the CE-LIF peak intensity of aptamer-ATP and ATP concentration in range of 10-700 μM, the regression equation was F=1.50+0.0470C(ATP) (R(2)=0.990), and the limit of detection was 1.28 μM (3S/N, n=5), which was one order magnitude lower than that of detection in solution by fluorescence method. The approach with excellent specificity and reproducibility has been successfully applied to detecting concentration of ATP in HeLa cell.

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

    PubMed

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

    2014-05-30

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

  20. Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner.

    PubMed

    Feng, Rui; Xu, Jianjun; Minobe, Etsuko; Kameyama, Asako; Yang, Lei; Yu, Lifeng; Hao, Liying; Kameyama, Masaki

    2014-05-01

    The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.

  1. The CoxD Protein, a Novel AAA+ ATPase Involved in Metal Cluster Assembly: Hydrolysis of Nucleotide-Triphosphates and Oligomerization

    PubMed Central

    Maisel, Tobias; Joseph, Stephanie; Mielke, Thorsten; Bürger, Jörg; Schwarzinger, Stephan; Meyer, Ortwin

    2012-01-01

    CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO2] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean KM value of 0.69±0.14 mM ATP and an apparent Vmax value of 19.3±2.3 nmol ATP hydrolyzed min−1 mg−1. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10–16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO2] cluster assembly are discussed. PMID:23077613

  2. Correlation between adenosine triphosphate levels, dopamine release and electrical activity in the carotid body: support for the metabolic hypothesis of chemoreception.

    PubMed

    Obeso, A; Almaraz, L; Gonzalez, C

    1985-11-25

    An unsolved issue for the arterial chemoreceptors is the mechanism by which hypoxia and other natural stimuli lead to an increase of activity in the carotid sinus nerve. According to the 'metabolic hypothesis', the hypoxic activation of the carotid body (CB) is mediated by a decrease of the ATP levels in the type I cells, which then release a neurotransmitter capable of exciting the sensory nerve endings. Using an in vitro preparation of cat CB, we report that ATP levels in the CB do in fact decrease when the organs are exposed to moderate, short lasting hypoxia (5 min 20% O2). Additionally, we found that decreases in ATP levels induced by 2-deoxyglucose (2 mM) or sodium cyanide (0.1 mM) are closely correlated with dopamine release from type I cells and electrical activity in the carotid sinus nerve elicited by these agents. The possible cause-effect relationship of these events is discussed.

  3. The mechanical integrity of healed diaphyseal bone defects grafted with calcium hydroxyapatite/calcium triphosphate ceramic in a new animal model.

    PubMed

    Black, R J; Zardiackas, L D; Teasdall, R; Hughes, J L

    1990-01-01

    The need for an animal model to test bone graft materials simulating a weight bearing clinical situation is identified. The concept, design and operative detail of a new model is described. This model involved the creation of a mid-diaphyseal wedge defect in the femur of the adult beagle which separated both cortices, plating with a six-hole dynamic compression plate, and allowed immediate full weight bearing. At six months plates were removed and immediate weight bearing was allowed for an additional six months to sacrifice. The initial animal project utilizing this model to evaluate a hydroxyapatite based synthetic graft material was performed using 12 dogs. In addition to the operative procedure, the retrieval testing in torsion of 12 healed grafted bones and their 12 contralateral unoperated controls is described and evaluated. Results showed no statistically significant difference between the torsional strength of test and control femurs (p less than or equal to 0.05). In addition, the future development of the model is discussed.

  4. Bis-Halogen-Anthraniloyl-Substituted Nucleoside 5′-Triphosphates as Potent and Selective Inhibitors of Bordetella pertussis Adenylyl Cyclase Toxin

    PubMed Central

    Geduhn, Jens; Dove, Stefan; Shen, Yuequan; Tang, Wei-Jen; König, Burkhard

    2011-01-01

    Whooping cough is caused by Bordetella pertussis and still constitutes one of the top five causes of death in young children, particularly in developing countries. The calmodulin-activated adenylyl cyclase (AC) toxin CyaA substantially contributes to disease development. Thus, potent and selective CyaA inhibitors would be valuable drugs for the treatment of whooping cough. However, it has been difficult to obtain potent CyaA inhibitors with selectivity relative to mammalian ACs. Selectivity is important for reducing potential toxic effects. In a previous study we serendipitously found that bis-methylanthraniloyl (bis-MANT)-IMP is a more potent CyaA inhibitor than MANT-IMP (Mol Pharmacol 72:526–535, 2007). These data prompted us to study the effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)-substituted nucleotides on CyaA and mammalian ACs. The novel nucleotides differentially inhibited CyaA and ACs 1, 2, and 5. Bis-ANT nucleotides inhibited CyaA competitively. Most strikingly, bis-Cl-ANT-ATP inhibited CyaA with a potency ≥100-fold higher than ACs 1, 2, and 5. In contrast to MANT-ATP, bis-MANT-ATP exhibited low intrinsic fluorescence, thereby substantially enhancing the signal-to noise ratio for the analysis of nucleotide binding to CyaA. The high sensitivity of the fluorescence assay revealed that bis-MANT-ATP binds to CyaA already in the absence of calmodulin. Molecular modeling showed that the catalytic site of CyaA is sufficiently spacious to accommodate both MANT substituents. Collectively, we have identified the first potent CyaA inhibitor with high selectivity relative to mammalian ACs. The fluorescence properties of bis-ANT nucleotides facilitate development of a high-throughput screening assay. PMID:20962032

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

    SciTech Connect

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

    1988-07-12

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

  6. A comparison of certain extracting agents for extraction of adenosine triphosphate (ATP) from microorganisms for use in the firefly luciferase ATP assay

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

    Firefly luciferase ATP assay is used in clinical and industrial applications, such as determination of urinary infection levels, microbial susceptibility testing, and monitoring of yeast levels in beverages. Three categories of extractants were investigated for their extracting efficiency. They were ionizing organic solvents, nonionizing organic solvents, and inorganic acids. Dimethylsulfoxide and formamide represented the ionizing organic solvents, while n-butanol, chloroform, ethanol, acetone, and methylene chloride were used for the nonionizing organic solvents. Nitric acid and perchloric acid were chosen for the inorganic acids category. Pathogens were tested with each solvent. They included: Saccharomyces carlsbergensis, E. coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter species, Proteus mirabilis, Proteus vulgaris, Staphylococcus epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, and Candida albicans. These results are shown in graphic representations.

  7. Application of the luciferin-luciferase enzyme system for determination of adenosine triphosphate (ATP) to studies on the mechanisms of herbicide action

    NASA Technical Reports Server (NTRS)

    St.john, J. B.

    1975-01-01

    The luciferin-luciferase enzyme system for determination of ATP is valuable for studies on the mechanisms of herbicide action. Investigations using this system have shown that certain herbicides may act by interfering with ATP production or by blocking ATP use, or by both mechanisms.

  8. Strain Promoted Click Chemistry of 2- or 8-Azidopurine and 5-Azidopyrimidine Nucleosides and 8-Azidoadenosine Triphosphate with Cyclooctynes. Application to Living Cell Fluorescent Imaging.

    PubMed

    Zayas, Jessica; Annoual, Marie; Das, Jayanta Kumar; Felty, Quentin; Gonzalez, Walter G; Miksovska, Jaroslava; Sharifai, Nima; Chiba, Akira; Wnuk, Stanislaw F

    2015-08-19

    Strain-promoted click chemistry of nucleosides and nucleotides with an azido group directly attached to the purine and pyrimidine rings with various cyclooctynes in aqueous solution at ambient temperature resulted in efficient formation (3 min to 3 h) of fluorescent, light-up, triazole products. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne, or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The 5-azidouridine and 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 min. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne in aqueous acetonitrile (3 h). The novel triazole adducts at the 2- or 8-position of adenine or 5-position of uracil rings induce fluorescence properties which were used for direct imaging in MCF-7 cancer cells without the need for traditional fluorogenic reporters. FLIM of the triazole click adducts demonstrated their potential utility for dynamic measuring and tracking of signaling events inside single living cancer cells.

  9. A Real-Time and Hands-On Research Course in Protein Purification and Characterization: Purification and Crystal Growth of Human Inosine Triphosphate Pyrophosphatase

    ERIC Educational Resources Information Center

    Kreiling, Jodi L.; Brader, Kerry; Kolar, Carol; Borgstahl, Gloria E. O.

    2011-01-01

    A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately…

  10. 2'-(R)-Fluorinated mC, hmC, fC and caC triphosphates are substrates for DNA polymerases and TET-enzymes.

    PubMed

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

    2016-12-13

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

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

    SciTech Connect

    Gavel, Olga Yu.; Kladova, Anna V.; Bursakov, Sergey A.; Dias, João M.; Texeira, Susana; Shnyrov, Valery L.; Moura, José J. G.; Moura, Isabel; Romão, Maria J.; Trincão, José

    2008-07-01

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

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

    PubMed

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

    2015-04-01

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

  13. Conservation of Complete Trimethylation of Lysine-43 in the Rotor Ring of c-Subunits of Metazoan Adenosine Triphosphate (ATP) Synthases*

    PubMed Central

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

    2015-01-01

    The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9–15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria. PMID:25608518

  14. Investigating the intermediates in the reaction of ribonucleoside triphosphate reductase from Lactobacillus leichmannii : An application of HF EPR-RFQ technology

    NASA Astrophysics Data System (ADS)

    Manzerova, Julia; Krymov, Vladimir; Gerfen, Gary J.

    2011-12-01

    In this investigation high-frequency electron paramagnetic resonance spectroscopy (HFEPR) in conjunction with innovative rapid freeze-quench (RFQ) technology is employed to study the exchange-coupled thiyl radical-cob(II)alamin system in ribonucleotide reductase from a prokaryote Lactobacillus leichmannii. The size of the exchange coupling ( Jex) and the values of the thiyl radical g tensor are refined, while confirming the previously determined (Gerfen et al. (1996) [20]) distance between the paramagnets. Conclusions relevant to ribonucleotide reductase catalysis and the architecture of the active site are presented. A key part of this work has been the development of a unique RFQ apparatus for the preparation of millisecond quench time RFQ samples which can be packed into small (0.5 mm ID) sample tubes used for CW and pulsed HFEPR - lack of this ability has heretofore precluded such studies. The technology is compatible with a broad range of spectroscopic techniques and can be readily adopted by other laboratories.

  15. Separation of adenosine diphosphate--adenosine triphosphate-exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase.

    PubMed

    Stahl, W L; Sattin, A; McIlwain, H

    1966-05-01

    1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.

  16. Potassium Aspartate Attenuates Brain Injury Induced by Controlled Cortical Impact in Rats Through Increasing Adenosine Triphosphate (ATP) Levels, Na+/K+-ATPase Activity and Reducing Brain Edema

    PubMed Central

    Gu, Yi; Zhang, Jie; Zhao, Yumei; Su, Yujin; Zhang, Yazhuo

    2016-01-01

    Background Potassium aspartate (PA), as an electrolyte supplement, is widely used in clinical practice. In our previous study, we found PA had neuroprotective effects against apoptosis after cerebral ischemia/reperfusion in rats. In this study, we examine whether PA has protective effects on traumatic brain injury (TBI). Material/Methods TBI was induced by controlled cortical impact (CCI) in rats. Vehicle treatment (control) or PA treatment was administered intraperitoneally at 30 minutes after CCI. The modified neurological severity score (mNSS) and cortical lesion volume were examined. Brain edema and blood-brain barrier (BBB) integrity were measured, as well as brain ATP contents, lactic acid levels, and Na+/K+-ATPase activities. Results We found that CCI induced cortical injury in rats. Acute PA treatment at the dose of 62.5 mg/kg and 125 mg/kg significantly improved neurological deficits (p<0.05 and p<0.001, respectively) and decreased the cortical lesion volume (p<0.05 and p<0.001, respectively) compared with vehicle-only treatment. PA treatment at the dose of 125 mg/kg attenuated brain edema and ameliorated BBB integrity. In addition, PA treatment significantly reduced the loss of ATP (p<0.01), reduced lactic acid levels (p<0.001), and increased the activity of Na+/K+-ATPase (p<0.01). Conclusions Our results indicate PA has neuroprotective effects on TBI through increasing ATP levels, Na+/K+-ATPase activity, and reducing brain edema. It provides experimental evidence for the clinical application of PA. PMID:27959885

  17. Membrane Fusion Protein Annexin 7: A Common Site of Action for Calcium, Guanosine Triphosphate, Protein Kinase C and Botulinum Toxin Type C in Regulated Exocytosis

    DTIC Science & Technology

    2002-01-01

    approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a calcium-dependent GTP-activated membrane fusion protein. In a...research project, we combined both in vitro and in vivo approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a...exocytotic membrane fusion process. The experimental strategies designed to test this hypothesis include the reconstituted membrane fusion system using

  18. Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

    PubMed Central

    Van de Werve, G; Hers, H G

    1979-01-01

    1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP. PMID:435271

  19. Nitrogenase of Klebsiella pneumoniae. Inhibition of acetylene reduction by magnesium ion explained by the formation of an inactive dimagnesium–adenosine triphosphate complex

    PubMed Central

    Thorneley, Roger N. F.; Willison, Keith R.

    1974-01-01

    Acetylene-reducing activity of purified nitrogenase from Klebsiella pneumoniae was studied over a range of ATP and Mg2+ concentrations at 15°C, pH7.8. Inhibition at Mg2+ concentrations of 2.5–30mm was due to the formation of the inactive complex, Mg2ATP. At higher Mg2+ concentrations an additional inhibitory effect was observed. The results were consistent with a MgATP complex being the active substrate with an apparent Km(MgATP)=0.4mm. PMID:4618775

  20. Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein.

    PubMed Central

    Mul, Y M; van Miltenburg, R T; De Clercq, E; van der Vliet, P C

    1989-01-01

    The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity. Images PMID:2587248

  1. Regulation of human norovirus VPg nucleotidylylation by ProPol and nucleoside triphosphate binding by its amino terminal sequence in vitro.

    PubMed

    Medvedev, Alexei; Viswanathan, Prasanth; May, Jared; Korba, Brent

    2017-03-01

    The VPg protein of human Norovirus (hNoV) is a multi-functional protein essential for virus replication. The un-cleaved viral precursor protein, ProPol (NS5-6) was 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific intracellular role for ProPol. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of VPg regulate its nucleotidylylation by ProPol. We provide evidence that VPg directly binds NTPs, inhibition of binding inhibits nucleotidylylation, and NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region inhibits nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation though several potential mechanisms.

  2. Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi alpha 2) and beta-adrenergic receptors in a porcine model of chronic episodic myocardial ischemia.

    PubMed Central

    Hammond, H K; Roth, D A; McKirnan, M D; Ping, P

    1993-01-01

    Regional myocardial ischemia is associated with increased levels of adenosine and norepinephrine, factors that may alter activation of the beta-adrenergic receptor (beta AR)-G protein-adenylyl cyclase pathway in the heart. We have used the ameroid constrictor model to determine whether alterations in myocardial signal transduction through the beta AR-G protein-adenylyl cyclase pathway occur in the setting of chronic episodes of reversible ischemia. Pigs were instrumented with ameroid occluders placed around the left circumflex coronary artery. 5 wk later, after ameroid closure, flow and function were normal in the ischemic bed, but flow (P = 0.001) and function (P < 0.03) were abnormal when metabolic demands were increased. The ischemic bed showed a reduction in myocardial beta AR number (P < 0.005). Despite regional downregulation of myocardial beta AR number, adenylyl cyclase activity was similar in the ischemic and control beds. Quantitative immunoblotting showed that the cardiac inhibitory GTP-binding protein, Gi alpha 2, was decreased in the ischemic bed (P = 0.02). In contrast, the cardiac stimulatory GTP-binding protein, Gs alpha, was increased in endocardial sections from the ischemic bed (P = < 0.05). Decreased Gi alpha 2 content was associated with decreased inhibition of adenylyl cyclase. Reduced Gi alpha 2 content, in conjunction with increased Gs alpha content in the endocardium, may provide a means by which adrenergic activation is maintained in the setting of chronic episodic myocardial ischemia. Images PMID:8254020

  3. A calcium-activated nucleotidase secreted from Ostertagia ostertagi fourth stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apyrases (ATP diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyze extracellular ATP and ADP to orthophosphate and AMP. Most apyrases have been structurally linked to the heat shock70/sugar kinase/actin superfamily which is associated with motility, adhesion, ...

  4. Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

    PubMed

    Kuby, S A; Hamada, M; Johnson, M S; Russell, G A; Manship, M; Palmieri, R H; Fleming, G; Bredt, D S; Mildvan, A S

    1989-08-01

    Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for the native enzyme) (Hamada and Kuby, 1978), and the K'd measured for Cr3+ ATP [corrected] and the synthetic peptide I1-45 (Fry et al., 1985b).

  5. Evaluation of Adenosine Triphosphate-Binding Cassette Transporter A1 (ABCA1) R219K and C-Reactive Protein Gene (CRP) +1059G/C Gene Polymorphisms in Susceptibility to Coronary Heart Disease.

    PubMed

    Li, Jing-Fang; Peng, Dian-Ying; Ling, Mei; Yin, Yong

    2016-08-25

    BACKGROUND This meta-analysis investigated the correlation of ABCA1 R219K and C-Reactive Protein Gene (CRP) +1059G/C gene polymorphisms with susceptibility to coronary heart disease (CHD). MATERIAL AND METHODS We searched PubMed, Springer link, Wiley, EBSCO, Ovid, Wanfang database, VIP database, and China National Knowledge Infrastructure (CNKI) databases to retrieve published studies by keyword. Searches were filtered using our stringent inclusion and exclusion criteria. Resultant high-quality data collected from the final selected studies were analyzed using Comprehensive Meta-analysis 2.0 software. Eleven case-control studies involving 3053 CHD patients and 3403 healthy controls met our inclusion criteria. Seven studies were conducted in Asian populations, 3 studies were done in Caucasian populations, and 1 was in an African population. RESULTS Our major finding was that ABCA1 R219K polymorphism increased susceptibility to CHD in allele model (OR=0.729, 95% CI=0.559~0.949, P=0.019) and dominant model (OR=0.698, 95% CI=0.507~0.961, P=0.027). By contrast, we were unable to find any significant association between the CRP +1059G/C polymorphism and susceptibility to CHD (allele model: OR=1.170, 95% CI=0.782~1.751, P=0.444; dominant model: OR=1.175, 95% CI=0.768~1.797, P=0.457). CONCLUSIONS This meta-analysis provides convincing evidence that polymorphism of ABCA1 R219K is associated with susceptibility to CHD while the CRP +1059G/C polymorphism appears to have no correlation with susceptibility to CHD.

  6. Diabetes and the control of pyruvate dehydrogenase in rat heart mitochondria by concentration ratios of adenosine triphosphate/adenosine diphosphate, of reduced/oxidized nicotinamide-adenine dinucleotide and of acetyl-coenzyme A/coenzyme A.

    PubMed Central

    Kerbey, A L; Radcliffe, P M; Randle, P J

    1977-01-01

    1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA. PMID:196589

  7. Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models.

    PubMed

    Real, R; González-Lobato, L; Baro, M F; Valbuena, S; de la Fuente, A; Prieto, J G; Alvarez, A I; Marques, M M; Merino, G

    2011-12-01

    In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.

  8. Brain Damage from Soman-Induced Seizures Is Greatly Exacerbated by Dimethyl sulfoxide (DMSO): Modest Neuroprotection by 2-Aminoethyl diphenylborinate (2- APB), a Transient Receptor Potential Channel Inhibitor and Inositol 1,4,5-triphosphate Receptor Antagonist

    DTIC Science & Technology

    2008-03-04

    dental cement. On the morning of the fifth or sixth day following surgeries, electrode-implanted animals were connected to an ECoG recording...epilepticus for several hours. Proconvulsive behavioral signs of soman intoxication included repetitive chewing , facial and forepaw clonus, motor...stereotypy, and wet- dog shakes. Overt motor convulsions were characterized by rhythmic clonic jerks of head and forepaws, rearing, salivation and Straub