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Sample records for adenosine diphosphate adp-induced

  1. Inhibition of adrenaline and adenosine diphosphate induced platelet aggregation by Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom.

    PubMed

    Lopez-Johnston, J C; de Bosch, N; Scannone, H; Rodríguez-Acosta, A

    2007-12-01

    The haemostatic components of venom from the genus Porthidium has been poorly studied, although it is known that severe manifestations occur when humans are envenomed, which include invasive oedema and disseminated ecchymosis. The effects of venom on blood platelets are commonly studied and are normally carried out with platelet-rich plasma (PRP). A series of crude venom dilutions was used to determine the effects of adenosine diphosphate (2 microM) and adrenaline (11 microM) induced platelet aggregation. Venom of Porthidium lansbergii hutmanni was fractioned by anionic exchange chromatography, and the fractions were also used to determine the 50% inhibition of adenosine diphosphate (ADP) and adrenaline-induced platelet aggregating dose (AD50). Crude venom has more effect in inhibiting adrenaline-induced aggregation (AD50 = 0.0043 microg) followed by the adenosine diphosphate (AD50 = 17 microg). Peaks I and II obtained by chromatography also inhibited adrenaline-induced platelet aggregation with an AD50 of 3.2 and 0.013 microg, respectively, and both peaks inhibited ADP-induced platelet aggregation with an AD50 of 10 microg. The main purpose of this work was to characterise the in vitro effects caused by P. lansbergii hutmanni crude venom and its fractions on the platelet aggregation mediated by adrenaline and ADP agonists. PMID:17891398

  2. Adenosine diphosphate-degrading activity in placenta.

    PubMed

    Barradas, M; Khokher, M; Hutton, R; Craft, I L; Dandona, P

    1983-02-01

    1. The degradation of ADP by the placenta and umbilical artery was investigated. 2. Supernatants from incubations of finely chopped placental and umbilical arterial tissue were incubated with [14C]ADP for various durations from 0 to 30 min. 3. Products of ADP degradation were separated by thin-layer chromatography and radioactivity incorporated into each product was measured. 4. Placental supernatants induced a more rapid degradation of ADP than the umbilical artery supernatants. The main product of ADP degradation by placental supernatants at 30 min was adenosine, whereas that of umbilical artery was AMP. 5. This conversion by placenta of ADP, a potent platelet aggregator and vasoconstrictor, into adenosine, a potent platelet anti-aggregator and vasodilator, may be important in the maintenance of perfusion of the foetoplacental unit. PMID:6822058

  3. Inhibition of poly(adenosine diphosphate-ribose) polymerase attenuates ventilator-induced lung injury

    PubMed Central

    Vaschetto, Rosanna; Kuiper, Jan W.; Chiang, Johnson; Haitsma, Jack J.; Juco, Jonathan W.; Uhlig, Stefan; Plötz, Frans B.; Della Corte, Francesco; Zhang, Haibo; Slutsky, Arthur S.

    2016-01-01

    Background Mechanical ventilation can induce organ injury associated with overwhelming inflammatory responses. Excessive activation of poly(adenosine diphosphate-ribose) polymerase enzyme following massive DNA damage may aggravate inflammatory responses. We thus hypothesized that the pharmacological inhibition of poly(adenosine diphosphate-ribose) polymerase by PJ-34 will attenuate ventilator-induced lung injury. Methods Anesthetized rats were subjected to intratracheal instillation of lipopolysaccharide at a dose of 6 mg/kg. The animals were then randomized to receive mechanical ventilation at either low tidal volume (6 mL/kg) with 5 cmH2O positive end-expiratory pressure or high tidal volume (15 mL/kg) with zero positive end-expiratory pressure, in the presence and absence of intravenous administration of PJ-34. Results The high tidal volume ventilation resulted in an increase in poly (adenosine diphosphate-ribose) polymerase activity in the lung. The treatment with PJ-34 maintained a greater oxygenation and a lower airway plateau pressure than the vehicle control group. This was associated with a decreased level of interleukin-6, active plasminogen activator inhibitor-1 in the lung, attenuated leukocyte lung transmigration and reduced pulmonary edema and apoptosis. The administration of PJ-34 also decreased the systemic levels of tumor necrosis factor-α and interleukin-6, and attenuated the degree of apoptosis in the kidney. Conclusion The pharmacological inhibition of poly(adenosine diphosphate-ribose) polymerase reduces ventilator-induced lung injury and protects kidney function. PMID:18212571

  4. Synthesis of the coenzymes adenosine diphosphate glucose, guanosine diphosphate glucose, and cytidine diphosphoethanolamine under primitive Earth conditions

    NASA Technical Reports Server (NTRS)

    Mar, A.; Oro, J.

    1991-01-01

    The nonenzymatic synthesis of the coenzymes adenosine diphosphate glucose (ADPG), guanosine diphosphate glucose (GDPG), and cytidine diphosphoethanolamine (CDP-ethanolamine) has been carried out under conditions considered to have been prevalent on the early Earth. The production of these compounds was performed by allowing simple precursor molecules to react under aqueous solutions, at moderate temperatures and short periods of time, with mediation by cyanamide or urea. These two condensing agents are considered to have been present in significant amounts on the primitive Earth and have been previously used in the nonenzymatic synthesis of several other important biochemical compounds. In our experiments, ADPG was obtained by heating glucose-1-phosphate (G1P) and ATP in the presence of cyanamide for 24 h at 70 degrees C. The reaction of G1P and GTP under the same conditions yielded GDPG. The cyanamide-mediated production of CDP-ethanolamine was carried out by reacting a mixture of ethanolamine phosphate and CTP for 24 h at 70 degrees C. The separation and identification of the reaction products was carried out by paper chromatography, thin-layer chromatography, high performance thin-layer chromatography, high performance liquid chromatography, both normal and reverse-phase, UV spectroscopy, enzymatic assays, and acid hydrolysis. Due to the mild conditions employed, and to the relative ease of these reactions, these studies offer a simple attractive system for the nonenzymatic synthesis of phosphorylated high-energy metabolic intermediates under conditions considered to have been prevalent on the ancient Earth.

  5. Clopidogrel and ticlopidine: P2Y12 adenosine diphosphate-receptor antagonists for the prevention of atherothrombosis.

    PubMed

    Savi, Pierre; Herbert, Jean-Marc

    2005-04-01

    Ticlopidine and clopidogrel belong to the same chemical family of thienopyridine adenosine diphosphate (ADP)-receptor antagonists. They have shown their efficacy as platelet antiaggregant and antithrombotic agents in many animal models, both ex vivo and in vivo. Although ticlopidine was discovered more than 30 years ago, it was only recently that the mechanism of action of ADP-receptor antagonists was characterized in detail. Ticlopidine and clopidogrel both behave in vivo as specific antagonists of P2Y (12), one of the ADP receptors on platelets. Metabolic steps that involve cytochrome P450-dependent pathways are required to generate the active metabolite responsible for this in vivo activity. The active moiety is a reactive thiol derivative that targets P2Y (12) on platelets. The interaction is irreversible, accounting for the observation that platelets are definitely antiaggregated, even if no active metabolite is detectable in plasma. The interaction is specific for P2Y (12); other purinoceptors such as P2Y (1) and P2Y (13) are spared. This results in inhibition of the binding of the P2Y (12) agonist 2-methylthio-ADP and the ADP-induced downregulation of adenylyl cyclase. Platelet aggregation is affected not only when triggered by ADP but also by aggregation inducers when used at concentrations requiring released ADP as an amplifier. The efficacy and safety of clopidogrel has been established in several large, randomized, controlled trials. The clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE) trial demonstrated the superiority of clopidogrel over acetylsalicylic acid (ASA) in patients at risk of ischemic events, including ischemic stroke, myocardial infarction (MI), and peripheral arterial disease. The clopidogrel in unstable angina to prevent recurrent ischemic events (CURE) trial showed a sustained, incremental benefit when clopidogrel was added to standard therapy (including ASA) in patients with unstable angina and non-Q-wave MI

  6. Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato mosaics.

    PubMed

    Boehlein, Susan K; Sewell, Aileen K; Cross, Joanna; Stewart, Jon D; Hannah, L Curtis

    2005-07-01

    Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors. PMID:15951484

  7. Hexokinase inhibitor screening based on adenosine 5'-diphosphate determination by electrophoretically mediated microanalysis.

    PubMed

    Wang, Tongdan; Kang, Jingwu

    2009-04-01

    A CE-based method for hexokinase inhibitor screening was developed in the present paper. In this method, hexokinase activity was assayed via electrophoretically mediated microanalysis (EMMA), which combines on-column hexokinase-mediated reaction and measurement of produced adenosine 5'-diphosphate (ADP) via electrophoretical separation and UV detection. Enzyme inhibition can be read out directly from the reduced peak area of ADP in comparison with a reference electropherogram obtained in the absence of any inhibitor. Conditions for on-column enzyme reaction and separation of adenosine 5'-triphosphate (ATP) and ADP were optimized. The optimal buffer composition for enzymatic reaction was 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl(2), whereas the optimal buffer composition for separation was 100 mM Tris-phosphate buffer (pH 5.5) containing 0.02% (m/v) hexadimethrine bromide (HDB). Fortunately, discontinuous buffer system can be adapted easily in the EMMA method. The time for separation was reduced dramatically to less than 3 min by reversing the direction of EOF via dynamically coating the capillary wall with the cationic polyelectrolyte HDB. Moreover, the peak tailing of ATP was also reduced by HDB coating. The Z' factor as high as 0.98 was obtained, indicating a high quality of the screening data. The present method is simple, robust and cost-effective. PMID:19306267

  8. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    PubMed

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A

    2008-05-15

    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation. PMID:18475194

  9. Inhibition of poly(adenosine diphosphate-ribose) polymerase using quinazolinone nucleus.

    PubMed

    Hemalatha, K; Madhumitha, G

    2016-09-01

    Poly(adenosine diphosphate-ribose) polymerase (PARP) is a group of enzymes with several subtypes and it manages various ailment such as cancer, inflammatory disorders, diabetes mellitus, neuronal injury, HIV infection, Parkinsonism, aging, and ischemia-reperfusion injury. Various PARP inhibitors share a common property of bicyclic lactam in its main structural frame. The core moiety containing bicyclic lactam rings are isoquinolinones, dihydroisoquinolinones, quinazolinediones, phthalazinones, quinazolinones, and phenanthridones. The quinazolinone with diverse substituents displayed low nanomolar inhibition. Quinazolinone is an important and vital molecule in the field of medicinal chemistry possessing multitude pharmacological actions. Though the chemistry of quinazolinones has been discussed through centuries, its concise role on PARP inhibition needed a special consideration. The aim of this review is to discover the effect of quinazolinone substitutents and its role in PARP inhibition. This precise review will discuss the effect of quinazolinones on PARP subtypes such as PARP-1, PARP-2, PARP-5a, and PARP-5b. In addition to its pharmacological actions, PARP inhibitors can also act as a chemosensitizing agent, and it is used in combination with the other anticancer agents. This summarization will definitely be a supportive report for the scientist working toward the novelty in the quinazolinone nucleus and its role in PARP inhibition. PMID:27470142

  10. Comparative pharmacokinetics and pharmacodynamics of platelet adenosine diphosphate receptor antagonists and their clinical implications.

    PubMed

    Floyd, Christopher N; Passacquale, Gabriella; Ferro, Albert

    2012-07-01

    Over the last two decades or more, anti-platelet therapy has become established as a cornerstone in the treatment of patients with ischaemic cardiovascular disease, since such drugs effectively reduce arterial thrombotic events. The original agent used in this context was aspirin (acetylsalicylic acid) but, with the advent of adenosine diphosphate (ADP) receptor antagonists, the use of dual anti-platelet therapy has resulted in further improvement in cardiovascular outcomes when compared with aspirin alone. The first group of platelet ADP receptor antagonists to be developed was the thienopyridine class, which comprise inactive pro-drugs that require in vivo metabolism to their active metabolites before exerting their inhibitory effect on the P2Y(12) receptor. Clopidogrel has been the principal ADP receptor antagonist in use over the past decade, but is limited by variability in its in vivo inhibition of platelet aggregation (IPA). The pharmacokinetics of clopidogrel are unpredictable due to their vulnerability to multiple independent factors including genetic polymorphisms. Expression of the 3435T/T genetic variant encoding the MDR1 gene for the P-glycoprotein efflux transporter results in a significantly reduced maximum drug concentration and area under the plasma concentration-time curve as intestinal absorption of clopidogrel is reduced; and the expression of the mutant *2 allele of CYP2C19 results in similar pharmacokinetic effects as the two cytochrome P450 (CYP)-mediated steps required for the production of the active metabolite of clopidogrel are impaired. These variable pharmacokinetics lead to erratic pharmacodynamics and cannot reliably be overcome with increased dosing. Both prasugrel, a third-generation thienopyridine, and ticagrelor, a cyto-pentyl-triazolo-pyrimidine, have more predictable pharmacokinetics and enhanced pharmacodynamics than clopidogrel. Neither appears to be affected by the same genetic polymorphisms as clopidogrel; prasugrel requires

  11. The status of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors in ovarian cancer, part 1: olaparib.

    PubMed

    Miller, Rowan E; Ledermann, Jonathan A

    2016-08-01

    Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors have shown promising clinical activity in epithelial ovarian cancer. Following the observation in vitro that PARP inhibition is synthetically lethal in tumors with BRCA mutations, PARP inhibition has become the first genotype-directed therapy for BRCA1- and BRCA2-associated ovarian cancer. However, it is becoming clear that PARP inhibition also may have clinical utility in cancers associated with defects or aberrations in DNA repair that are unrelated to BRCA mutations. Deficient DNA repair mechanisms are present in approximately 30% to 50% of high-grade serous ovarian cancers, the most common histologic subtype. Olaparib is the best-studied PARP inhibitor to date, and a number of phase 3 trials with this agent are underway. This article reviews the development of olaparib for ovarian cancer and discusses the current evidence for its use, ongoing studies, future research directions, and the challenges ahead. PMID:27487106

  12. Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

    PubMed Central

    Peng, Qian-Yi; Zou, Yu; Zhang, Li-Na; Ai, Mei-Lin; Liu, Wei; Ai, Yu-Hang

    2016-01-01

    Background: Acute lung injury (ALI) is a common complication of sepsis that is associated with high mortality. Intracellular Ca2+ overload plays an important role in the pathophysiology of sepsis-induced ALI, and cyclic adenosine diphosphate ribose (cADPR) is an important regulator of intracellular Ca2+ mobilization. The cluster of differentiation 38 (CD38)/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALI is still unknown. This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALI and whether blocking cADPR-mediated calcium overload attenuates ALI. Methods: Septic rat models were established by cecal ligation and puncture (CLP). Rats were divided into the sham group, the CLP group, and the CLP+ 8-bromo-cyclic adenosine diphosphate ribose (8-Br-cADPR) group. Nicotinamide adenine dinucleotide (NAD+), cADPR, CD38, and intracellular Ca2+ levels in the lung tissues were measured at 6, 12, 24, and 48 h after CLP surgery. Lung histologic injury, tumor necrosis factor (TNF)-α, malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activities were measured. Results: NAD+, cADPR, CD38, and intracellular Ca2+ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery. Treatment with 8-Br-cADPR, a specific inhibitor of cADPR, significantly reduced intracellular Ca2+ levels (P = 0.007), attenuated lung histological injury (P = 0.023), reduced TNF-α and MDA levels (P < 0.001 and P = 0.002, respectively) and recovered SOD activity (P = 0.031) in the lungs of septic rats. Conclusions: The CD38/cADPR pathway is activated in the lungs of septic rats, and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALI. PMID:27411462

  13. Adenosine diphosphate restricts the protein remodeling activity of the Hsp104 chaperone to Hsp70 assisted disaggregation

    PubMed Central

    Kłosowska, Agnieszka; Chamera, Tomasz; Liberek, Krzysztof

    2016-01-01

    Hsp104 disaggregase provides thermotolerance in yeast by recovering proteins from aggregates in cooperation with the Hsp70 chaperone. Protein disaggregation involves polypeptide extraction from aggregates and its translocation through the central channel of the Hsp104 hexamer. This process relies on adenosine triphosphate (ATP) hydrolysis. Considering that Hsp104 is characterized by low affinity towards ATP and is strongly inhibited by adenosine diphosphate (ADP), we asked how Hsp104 functions at the physiological levels of adenine nucleotides. We demonstrate that physiological levels of ADP highly limit Hsp104 activity. This inhibition, however, is moderated by the Hsp70 chaperone, which allows efficient disaggregation by supporting Hsp104 binding to aggregates but not to non-aggregated, disordered protein substrates. Our results point to an additional level of Hsp104 regulation by Hsp70, which restricts the potentially toxic protein unfolding activity of Hsp104 to the disaggregation process, providing the yeast protein-recovery system with substrate specificity and efficiency in ATP consumption. DOI: http://dx.doi.org/10.7554/eLife.15159.001 PMID:27223323

  14. Online cleanup of accelerated solvent extractions for determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly using high-performance liquid chromatography.

    PubMed

    Xue, Xiaofeng; Wang, Feng; Zhou, Jinhui; Chen, Fang; Li, Yi; Zhao, Jing

    2009-06-10

    Determination of the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly is important for the study of its pharmacological activities, health benefits, and adenosine phosphate degradation. In this study was developed a novel method to determine ATP, ADP, and AMP levels in royal jelly using accelerated solvent extraction (ASE) followed by online cleanup and high-performance liquid chromatography (HPLC) with diode array detection (DAD). The optimum extraction conditions were obtained using an 11 mL ASE cell, ethanol/water (5:5 v/v) as the extraction solvent, 1500 psi, 80 degrees C, a 5 min static time, and a 60% flush volume. Optimum separation of the three compounds was achieved in <25 min using a Waters XBridge Shield RP18 column with 0.05 mol L(-1) NH(4)H(2)PO(4) (pH 5.70) and acetonitrile as the mobile phase. Detection was performed at 257 nm. The method was sensitive (LOD adenosine phosphate extraction procedures (perchloric acid). The results indicate that the two techniques are similar in terms of recovery and reproducibility, but when other factors such as extraction time, environmental protection, and worker's health are considered, ASE is preferable to the classical extraction method. With this ASE-HPLC method, a minisurvey of ATP, ADP, and AMP levels in 15 samples of royal jelly of different origins was performed. Sample results indicated that the AMP concentration was 24.2-2214.4 mg kg(-1), whereas ATP and ADP were not detectable or present only at low levels. PMID:19435312

  15. Impact of aspirin dose on adenosine diphosphate-mediated platelet activities. Results of an in vitro pilot investigation.

    PubMed

    Tello-Montoliu, Antonio; Thano, Estela; Rollini, Fabiana; Patel, Ronakkumar; Wilson, Ryan E; Muñiz-Lozano, Ana; Franchi, Francesco; Darlington, Andrew; Desai, Bhaloo; Guzman, Luis A; Bass, Theodore A; Angiolillo, Dominick J

    2013-10-01

    Different aspirin dosing regimens have been suggested to impact outcomes when used in combination with adenosine diphosphate (ADP) P2Y12 receptor antagonists. Prior investigations have shown that not only aspirin, but also potent ADP P2Y12 receptor blockade can inhibit thromboxane A2-mediated platelet activation. The impact of aspirin dosing on ADP mediated platelet activities is unknown and represents the aim of this in vitro pilot pharmacodynamic (PD) investigation. Twenty-six patients with stable coronary artery disease on aspirin 81 mg/day and P2Y12 naïve were enrolled. PD assessments were performed at baseline, while patients were on 81 mg/day aspirin and after switching to 325 mg/day for 7 ± 2 days with and without escalating concentrations (vehicle, 1, 3, and 10 μM) of prasugrel's active metabolite (P-AM). PD assays included flow cytometric assessment of VASP to define the platelet reactivity index (PRI) and the Multiplate Analyzer (MEA) using multiple agonists [ADP, ADP + prostaglandin (PGE1), arachidonic acid (AA), and collagen]. Escalating P-AM concentrations showed incremental platelet P2Y12 inhibition measured by VASP-PRI (p<0.001). However, there were no differences according to aspirin dosing regimen at any P-AM concentration (vehicle: p=0.899; 1 μM: p=0.888; 3 μM: p=0.524; 10 μM: p=0.548). Similar findings were observed in purinergic markers assessed by MEA (ADP and ADP+PGE1). P-AM addition significantly reduced AA and collagen induced platelet aggregation (p<0.001 for all measures), irrespective of aspirin dose. In conclusion, aspirin dosing does not appear to affect PD measures of ADP-mediated platelet reactivity irrespective of the degree of P2Y12 receptor blockade. P2Y12 receptor blockade modulates platelet reactivity mediated by alternative activators. PMID:23884248

  16. Traumatic brain injury causes platelet adenosine diphosphate and arachidonic acid receptor inhibition independent of hemorrhagic shock in humans and rats

    PubMed Central

    Castellino, Francis J.; Chapman, Michael P.; Donahue, Deborah L.; Thomas, Scott; Moore, Ernest E.; Wohlauer, Max V.; Fritz, Braxton; Yount, Robert; Ploplis, Victoria; Davis, Patrick; Evans, Edward; Walsh, Mark

    2014-01-01

    BACKGROUND Coagulopathy in traumatic brain injury (CTBI) is a well-established phenomenon, but its mechanism is poorly understood. Various studies implicate protein C activation related to the global insult of hemorrhagic shock or brain tissue factor release with resultant platelet dysfunction and depletion of coagulation factors. We hypothesized that the platelet dysfunction of CTBI is a distinct phenomenon from the coagulopathy following hemorrhagic shock. METHODS We used thrombelastography with platelet mapping as a measure of platelet function, assessing the degree of inhibition of the adenosine diphosphate (ADP) and arachidonic acid (AA) receptor pathways. First, we studied the early effect of TBI on platelet inhibition by performing thrombelastography with platelet mapping on rats. We then conducted an analysis of admission blood samples from trauma patients with isolated head injury (n = 70). Patients in shock or on clopidogrel or aspirin were excluded. RESULTS In rats, ADP receptor inhibition at 15 minutes after injury was 77.6% ± 6.7% versus 39.0% ± 5.3% for controls (p < 0.0001). Humans with severe TBI (Glasgow Coma Scale [GCS] score ≤ 8) showed an increase in ADP receptor inhibition at 93.1% (interquartile range [IQR], 44.8–98.3%; n = 29) compared with 56.5% (IQR, 35–79.1%; n = 41) in milder TBI and 15.5% (IQR, 13.2–29.1%) in controls (p = 0.0014 and p < 0.0001, respectively). No patient had significant hypotension or acidosis. Parallel trends were noted in AA receptor inhibition. CONCLUSION Platelet ADP and AA receptor inhibition is a prominent early feature of CTBI in humans and rats and is linked to the severity of brain injury in patients with isolated head trauma. This phenomenon is observed in the absence of hemorrhagic shock or multisystem injury. Thus, TBI alone is shown to be sufficient to induce a profound platelet dysfunction. (J Trauma Acute Care Surg. 2014;76: 1169–1176. PMID:24747445

  17. Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

    PubMed

    Olivier, Christoph B; Weik, Patrick; Meyer, Melanie; Weber, Susanne; Diehl, Philipp; Bode, Christoph; Moser, Martin; Zhou, Qian

    2016-08-01

    Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA

  18. Plant extracts inhibit ADP-induced platelet activation in humans: their potential therapeutic role as ADP antagonists.

    PubMed

    Jagroop, Indera Anita

    2014-01-01

    Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. Platelet hyperactivity is associated with vascular disease and also has a key role in haemostasis and thrombosis. ADP activates platelets through three purinoceptor subtypes, the G(q)-coupled P2Y(1) receptor, G(i)-coupled P2Y(12) receptor and P2X(1) ligand-gated cation channel. Platelet ADP purinergic receptors are therefore suitable targets for antiplatelet drugs. Thienopyridines such as clopidogrel and ticlopidine, as well as other ADP receptor antagonists like prasugrel, ticagrelor, cangrelor and elinogrel have demonstrated clinical benefits via the inhibition of the selective purinergic ADP receptor, P2Y(12). However, they still have limitations in their mode of action and efficacy, like increased risk of bleeding. Thus, the ongoing pursuit to develop newer and more effective antiplatelet agents continues. There is a growing interest in the purinergic antiplatelet properties exhibited by plant extracts. This article considers the following: pomolic acid isolated from Licania pittieri, brazilin isolated from the heartwood of Caesalpinia sappan L, phylligenin isolated from the twigs of Muraltia vulpina, bark oil of Gonystylus velutinus, seed and bark extracts from Aesculus hippocastanum L. and red wine phenolics and catechins isolated from green tea. Moreover, the method used to investigate platelet purinergic receptors should be considered, since using a more sensitive, high-resolution platelet sizer can sometimes detect platelet variations when the light transmission method was not able to do so. The exact mechanisms by which these plant extracts work need further investigation. They all however inhibit ADP-induced activation in human platelets. This could explain, at least in part, the protective effect of plant extracts as antiplatelet agents. PMID:24190032

  19. Roles of glia limitans astrocytes and CO in ADP-induced pial arteriolar dilation in newborn pigs

    PubMed Central

    Kanu, Alie; Leffler, Charles W.

    2009-01-01

    Background and Purpose Astrocytes, neurons, and microvessels together form a neurovascular unit allowing blood flow to match neuronal activity. Adenosine diphosphate (ADP) is an important signaling molecule in the brain, and dilation in response to ADP is astrocyte-dependent in rats and newborn pigs. Carbon monoxide (CO), produced endogenously by catabolism of heme to CO, iron, and biliverdin via heme oxygenase (HO), is an important cell signaling molecule in the neonatal cerebral circulation. We hypothesize ADP stimulates CO production by glia limitans astrocytes and that this CO causes pial arteriolar dilation. Methods Experiments were performed using anesthetized piglet with closed cranial windows, and freshly isolated piglet astrocytes and microvessels. Astrocyte injury was caused by topical application of L-2-alpha aminoadipic acid (2 mM, 5 h). Cerebrospinal fluid (CSF) was collected from under the cranial windows for measurement of ADP-stimulated CO production. CO was measured by gas chromatography-mass spectroscopy analysis. Results Before, but not after, astrocyte injury in vivo, topical ADP stimulated both CO production and dilation of pial arterioles. Astrocyte injury did not block dilation to isoproterenol or bradykinin. Chromium mesoporphyrin, an inhibitor of HO, also prevented the ADP-induced increase in CSF CO and pial arteriolar dilation caused by ADP but not dilation to sodium nitroprusside. ADP also increased CO production by freshly isolated piglet astrocytes and cerebral microvessels, although the increase was smaller in the microvessels. Conclusions These data suggest that glia limitans astrocytes employ CO as a gasotransmitter to cause pial arteriolar dilation in response to ADP. PMID:19164779

  20. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

    PubMed Central

    Bell, D N; Spain, S; Goldsmith, H L

    1989-01-01

    A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone. PMID:2605298

  1. Microwave-Assisted Hydrothermal Rapid Synthesis of Amorphous Calcium Phosphate Mesoporous Microspheres Using Adenosine 5'-Diphosphate and Application in pH-Responsive Drug Delivery.

    PubMed

    Qi, Chao; Zhu, Ying-Jie; Sun, Tuan-Wei; Wu, Jin; Chen, Feng

    2015-11-01

    Herein we report a rapid and green strategy for the preparation of amorphous calcium phosphate mesoporous microspheres (ACP-MSs) using adenosine 5'-diphosphate disodium salt (ADP) as an organic phosphorus source by a microwave-assisted hydrothermal method. The effects of the pH value, the reaction time, and temperature on the crystal phase and morphology of the product are investigated. The ADP biomolecules used in this strategy play an important role in the formation of ACP-MSs. The as-prepared ACP-MSs are efficient for anticancer drug delivery by using doxorubicin (Dox) as a model drug, and the Dox-loaded ACP-MSs show a high ability to damage cancer cells. Moreover, the ACP-MSs drug delivery system exhibits a pH-responsive drug-release behavior due to the degradation of ACP-MSs at a low pH value, thus, it is promising for applications in pH-responsive drug delivery. PMID:26248600

  2. Crystal structures of human sulfotransferases SULT1B1 and SULT1C1 complexed with the cofactor product adenosine-3'- 5'-diphosphate (PAP)

    SciTech Connect

    Dombrovski, Luidmila; Dong, Aiping; Bochkarev, Alexey; Plotnikov, Alexander N.

    2008-09-17

    Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3{prime}-phosphoadenosine 5{prime}-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites. Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1). Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols. Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver. SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown. We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3{prime}-5{prime}-diphosphate (PAP).

  3. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    SciTech Connect

    Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A.; Solomon, Benjamin

    2014-02-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.

  4. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots.

    PubMed

    Chung, Tze-Wen; Lin, Pei-Yi; Wang, Shoei-Shen; Chen, Yen-Fung

    2014-01-01

    Chitosan nanoparticles (NPs) decorated with adenosine diphosphate (ADP) (ANPs) or fibrinogen (FNPs) were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP) or FNP (clot/FNP) were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1 ± 14.0, 251.0 ± 9.8, and 326.5 ± 14.5 nm and zeta potentials of 24.1 ± 0.5, 20.6 ± 1.9, and 15.3 ± 1.5 mV (n=4), respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR) spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM) micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP), clot/ANP, and clot/FNP (at 0.05 wt%) were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc) of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM), were significantly (P<0.05) (n=4) shorter than that of a clot induced by a phosphate-buffered solution (PBS) (clot/PBS) (63.6% ± 3.1%, 48.3% ± 6.2%, and 63.2% ± 4.7%, respectively). The ΔF2 values in the spectra of frequency shifts associated with the propagation of fibrin networks in the clot/ANPs and clot/FNPs were significantly lower than those of clot/PBS. Interestingly, texture profile analysis of the compressional properties showed significantly lower hardness and compressibility in clot/NPs and clot/ANPs (P<0.05 or better) (n=4) compared with

  5. Pharmacokinetic study of adenosine diphosphate-encapsulated liposomes coated with fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute in an anticancer drug-induced thrombocytopenia rat model.

    PubMed

    Taguchi, Kazuaki; Ujihira, Hayato; Watanabe, Hiroshi; Fujiyama, Atsushi; Doi, Mami; Takeoka, Shinji; Ikeda, Yasuo; Handa, Makoto; Otagiri, Masaki; Maruyama, Toru

    2013-10-01

    A fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, adenosine diphosphate (ADP)-encapsulated liposome [H12-(ADP)-liposome] was designed to achieve optimal performance as a homeostatic agent and expected as a synthetic platelet alternative. For the purpose of efficient function as platelet substitute, H12-(ADP)-liposomes should potentially have both acceptable pharmacokinetic and biodegradable properties under conditions of an adaptation disease including thrombocytopenia induced by anticancer drugs. The aim of this study was to characterize the pharmacokinetics of H12-(ADP)-liposomes in busulphan-induced thrombocytopenic rats using (14) C, (3) H double radiolabeled H12-(ADP)-liposomes, in which the encapsulated ADP and liposomal membrane (cholesterol) were labeled with (14) C and (3) H, respectively. After the administration of H12-(ADP)-liposomes, they were determined to be mainly distributed to the liver and spleen and disappeared from organs within 7 days after injection. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane (cholesterol) was mainly eliminated in feces. The successive dispositions of the H12-(ADP)-liposomes were similar in both normal and thrombocytopenic rats. However, the kinetics of H12-(ADP)-liposomes in thrombocytopenic rats was more rapid, compared with the corresponding values for normal rats. These findings, which well reflect the clinical features of patients with anticancer drug-induced thrombocytopenia, provide useful information for the development of the H12-(ADP)-liposomes for future clinical use. PMID:23918456

  6. Inhalation of nitric oxide inhibits ADP-induced platelet aggregation and alpha-granule release.

    PubMed

    Hagberg, I A; Sølvik, U Ø; Opdahl, H; Roald, H E; Lyberg, T

    1999-01-01

    To gather further information about the effects on blood platelet activation of in vivo exposure to nitric oxide (NO), platelet reactivity was studied in blood from healthy, non-smoking male volunteers before and after 30 min inhalation of 40 ppm NO. Whole blood was stimulated in vitro with adenosine diphosphate or thrombin receptor activation peptide (TRAP-6). In an ex vivo perfusion model, non-anticoagulated blood was exposed to immobilised collagen at arterial blood flow conditions (2600 s(-1)). Blood samples from both the in vitro and ex vivo experiments were stained with fluorochrome-labelled Annexin-V and antibodies against CD42a, CD45, CD49b, CD61, CD62P and fibrinogen, and analysed with a three-colour flow cytometry technique. NO inhalation reduced the platelet activation response to adenosine diphosphate (ADP) stimulation by decreasing platelet-platelet aggregation, alpha-granule release and platelet-leukocyte conjugate formation. TRAP-stimulated platelet activation, collagen-induced platelet activation and thrombus growth was unaffected by NO inhalation. We therefore suggest an ADP receptor inhibitor mode of action of inhaled NO, selective on the newly suggested G protein- and phospholipase C-coupled P2Y1 receptor. Our results demonstrate that blood platelet activation in healthy subjects is modulated by inhalation of NO in therapeutically relevant doses, although the clinical impact of our findings remains unclear. PMID:16801117

  7. Structure of the nucleotide-binding subunit B of the energy producer A1A0 ATP synthase in complex with adenosine diphosphate.

    PubMed

    Kumar, Anil; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

    2008-11-01

    A1A0 ATP synthases are the major energy producers in archaea. Like the related prokaryotic and eukaryotic F1F0 ATP synthases, they are responsible for most of the synthesis of adenosine triphosphate. The catalytic events of A1A0 ATP synthases take place inside the A3B3 hexamer of the A1 domain. Recently, the crystallographic structure of the nucleotide-free subunit B of Methanosarcina mazei Gö1 A1A0 ATP synthase has been determined at 1.5 A resolution. To understand more about the nucleotide-binding mechanism, a protocol has been developed to crystallize the subunit B-ADP complex. The crystallographic structure of this complex has been solved at 2.7 A resolution. The ADP occupies a position between the essential phosphate-binding loop and amino-acid residue Phe149, which are involved in the binding of the antibiotic efrapeptin in the related F1F0 ATP synthases. This trapped ADP location is about 13 A distant from its final binding site and is therefore called the transition ADP-binding position. In the trapped ADP position the structure of subunit B adopts a different conformation, mainly in its C-terminal domain and also in the final nucleotide-binding site of the central alphabeta-domain. This atomic model provides insight into how the substrate enters into the nucleotide-binding protein and thereby into the catalytic A3B3 domain. PMID:19020348

  8. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

    Mills, G C; Schmalstieg, F C; Trimmer, K B; Goldman, A S; Goldblum, R M

    1976-01-01

    Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides. PMID:1066699

  9. 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.

  10. Dilithium barium diphosphate.

    PubMed

    Dridi, Nezha; Arbib, E; Boukhari, Ali; Holt, Elizabeth M

    2002-06-01

    The crystal structure of the novel title diphosphate, Li(2)BaP(2)O(7), exists with a three-dimensional lattice composed of BaO(9) polyhedra linked to corner- and edge-sharing P(2)O(7) diphosphate groups, forming layers parallel to the (010) plane, the layers being linked by P[bond]O[bond]Ba bridges. Tunnels thus created between the layers are occupied by Li(+) cations, two of which lie on twofold axes. PMID:12050405

  11. Geranyl diphosphate synthase from mint

    SciTech Connect

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  12. Geranyl diphosphate synthase from mint

    SciTech Connect

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  13. Inhibition of Primary ADP-Induced Platelet Aggregation in Normal Subjects after Administration of Nitrofurantoin (Furadantin)

    PubMed Central

    Rossi, Ennio C.; Levin, Nathan W.

    1973-01-01

    The evidence indicating that platelets may play a role in the occurrence of certain thromboembolic phenomena has stimulated a search for inhibitors of platelet function. This report presents data to indicate that nitrofurantoin is a potent inhibitor of primary ADP-induced platelet aggregation. The addition of 10 μM nitrofurantoin to citrated platelet-rich plasma obtained from 12 normal subjects produced a 29±6% (2 SD) inhibition of the velocity of platelet aggregation induced by 2 μM ADP. The inhibitory effect of nitrofurantoin demonstrated competitive kinetics in respect to ADP. The intravenous (180 mg) or oral (200 mg) administration of nitrofurantoin produced a serum nitrofurantoin concentration ranging from 2.7 to 23 μM in 28 normal subjects. Platelet-rich plasma obtained from these subjects demonstrated inhibition of the velocity of ADP-induced platelet aggregation that correlated with the log of the serum nitrofurantoin concentration (P < 0.001). Collagen-induced platelet aggregation was also inhibited in a dose-related manner, and the bleeding time was significantly prolonged in the two subjects with the highest serum nitrofurantoin concentration. These studies indicate that nitrofurantoin in vivo inhibits platelet function to a degree that is proportional to the serum nitrofurantoin concentration. PMID:4729043

  14. The preparation of adenosine 5′-pyrophosphate by a non-enzymic method

    PubMed Central

    Dawson, R. M. C.; Ford, M.; Eichberg, J.

    1965-01-01

    1. A non-enzymic method for the preparation of adenosine 5′-diphosphate is described, in which the terminal phosphate of adenosine 5′-triphosphate is transferred to methanol in the presence of hydrochloric acid. The final purified product can be obtained in 60% yield. 2. Experiments with [14C]methanol showed that no methylation of the adenosine diphosphate occurs during the reaction. 3. Confirmation that the pyrophosphate moiety of the adenosine diphosphate produced was in the 5′-position was obtained by: (a) periodate oxidation; (b) treatment with apyrase and examination of the resulting adenylic acid isomer by paper chromatography. 4. The method appears to be generally applicable to the preparation of nucleoside 5′-diphosphates from the corresponding nucleoside 5′-triphosphates. PMID:14333545

  15. Methodological considerations for the assessment of ADP induced platelet aggregation using the Multiplate® analyser.

    PubMed

    Johnston, Lisa R; Larsen, Peter D; La Flamme, Anne C; Harding, Scott A

    2013-01-01

    Factors affecting the Multiplate® assay's analytical precision have not been well defined. We investigated the effect of methodological factors on the measurement of ADP-induced platelet aggregation using the Multiplate® assay. ADP-induced platelet aggregation was analysed in whole blood using the Multiplate® assay. We tested the reproducibility of measurement, the effect of different anticoagulants (hirudin, citrate and heparin) and the effect of time delay (15, 30, 45, 60, 120 and 180 minutes) between sampling and analysis in patients. The use of a manual calibrated pipette with the Multiplate® analyser was also tested. The mean coefficient of variation (CV) using the manufacturers recommended methods was 10.8 ± 8.7% (n = 30). When compared to hirudin (359.5 ± 309 AU*min) the use of heparin (521.0 ± 316 AU*min, p = 0.0015) increased platelet aggregation, while the use of sodium citrate (245.0 ± 209 AU*min, p = 0.003) decreased the platelet aggregation (n = 20). The addition of CaCl2 to the citrate-anticoagulated blood resulted in platelet aggregation levels similar to hirudin. Platelet aggregation varied with time delay (n = 20). When compared to platelet aggregation at 30 minutes (391.1 ± 283 AU*min), platelet aggregation was reduced at 60 minutes (335.2 ± 251.6 AU*min, p < 0.05), 120 minutes (198.8 ± 122.9 AU*min, p < 0.001) and 180 minutes (160.7 ± 92 AU*min, p < 0.001). The use of a manual calibrated pipette did not significantly reduce the mean CV in the assay (n = 20). Methodological factors such as the anticoagulant used and the time delay should be standardised where possible to reduce variability, and allow thresholds derived from one study to be comparable across multiple studies. PMID:22686487

  16. Interaction of salvianolic acids and notoginsengnosides in inhibition of ADP-induced platelet aggregation.

    PubMed

    Yao, Yan; Wu, Wan-Ying; Liu, Ai-Hua; Deng, Shao-Sheng; Bi, Kai-Shun; Liu, Xuan; Guo, De-An

    2008-01-01

    Salvia miltiorrhiza and Panax notoginseng were both considered to be beneficial to cardiovascular diseases in traditional Chinese medicine and often used in combination. To examine the possible interaction between them, the effects of the active fractions of these two herbs, salvianolic acids (SA) and notoginsengnosides (NG), on platelet aggregation were checked respectively or in combination in vitro and in vivo. Both the platelet aggregation of platelet rich plasma (PRP) and washed platelet after ADP induction were checked. In vitro study showed that both SA and NG had an inhibitory effect on platelet aggregation. However, there is no synergistic effect of the combination of SA and NG in vitro. In vivo study showed that i.g. 550 mg/kg/day SA or NG for 5 days could significantly inhibit ADP-induced platelet aggregation of PRP. Moreover, combination of SA and NG at a ratio of 5:1 had a synergistic effect on platelet aggregation of PRP. The mechanism for the synergism of SA and NG in vivo was not clear. High performance liquid chromatography analysis of the plasma of rats received SA, NG or combination of SA and NG showed that co-administration of NG caused change in the plasma distribution profile of SA. The influence of combination on the absorption and/or metabolism of SA may be one of the reasons for the synergism of SA and NG in vivo. PMID:18457363

  17. Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

    NASA Astrophysics Data System (ADS)

    Wei, Yuxi; Wang, Changyun; Li, Jing; Guo, Qi; Qi, Hongtao

    2009-09-01

    We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA ( P<0.01) and increasing the synthesis of 6-keto-PGF1α, thus changing the plasma TXB2/6-keto-PGF1α balance when the platelets were activated ( P<0.01). Therefore, STP altered AA metabolism and these findings partly revealed the molecular mechanism by which STP inhibits ADP-induced platelet aggregation.

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

    PubMed

    Koziołkiewicz, W; Pankowski, J; Janecka, A

    1978-01-01

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

  19. Adenosine transporters.

    PubMed

    Thorn, J A; Jarvis, S M

    1996-06-01

    1. In mammals, nucleoside transport is an important determinant of the pharmacokinetics, plasma and tissue concentration, disposition and in vivo biological activity of adenosine as well as nucleoside analogues used in antiviral and anticancer therapies. 2. Two broad types of adenosine transporter exist, facilitated-diffusion carriers and active processes driven by the transmembrane sodium gradient. 3. Facilitated-diffusion adenosine carriers may be sensitive (es) or insensitive (ei) to nanomolar concentrations of the transport inhibitor nitrobenzylthioinosine (NBMPR). Dipyridamole, dilazep and lidoflazine analogues are also more potent inhibitors of the es carrier than the ei transporter in cells other than those derived from rat tissues. 4. The es transporter has a broad substrate specificity (apparent Km for adenosine approximately 25 microM in many cells at 25 degrees C), is a glycoprotein with an average apparent Mr of 57,000 in human erythrocytes that has been purified to near homogeneity and may exist in situ as a dimer. However, there is increasing evidence to suggest the presence of isoforms of the es transporter in different cells and species, based on kinetic and molecular properties. 5. The ei transporter also has a broad substrate specificity with a lower affinity for some nucleoside permeants than the es carrier, is genetically distinct from es but little information exists as to the molecular properties of the protein. 6. Sodium-dependent adenosine transport is present in many cell types and catalysed by four distinct systems, N1-N4, distinguished by substrate specificity, sodium coupling and tissue distribution. 7. Two genes have been identified which encode sodium-dependent adenosine transport proteins, SNST1 from the sodium/glucose cotransporter (SGLT1) gene family and the rat intestinal N2 transporter (cNT1) from a novel gene family including a bacterial nucleoside carrier (NupC). Transcripts of cNT1, which encodes a 648-residue protein, are

  20. Quantification of adenosine triphosphate, adenosine diphosphate, and creatine phosphate in sterlet spermatozoa during maturation.

    PubMed

    Fedorov, P; Dzyuba, B; Fedorova, G; Grabic, R; Cosson, J; Rodina, M

    2015-11-01

    Sturgeon spermatozoa maturation during their passage through the kidney is a prerequisite for initiation of motility. Samples of sterlet () testicular sperm (TS) were matured in vitro by incubation in seminal fluid (SF) or in SF supplemented with carbonyl cyanide -chlorophenyl hydrazone (CCCP; a respiration uncoupling agent). Sperm was diluted in activation medium (AM) containing 10 m Tris-HCl buffer (pH 8.5) and 0.25% Pluronic, and spermatozoon motility was assessed. Samples were taken and fixed in 3 perchloric acid at 3 points in the incubation process. Quantification of ATP, ADP, and creatine phosphate (CrP) was conducted using liquid chromatography/high-resolution mass spectrometry. We observed a significant decrease in CrP during artificial maturation of TS in SF. In contrast, ATP and ADP were not significantly affected. Addition of CCCP to SF halted maturation and led to significantly lower CrP whereas ADP significantly increased and ATP was unaffected. Dilution of matured and immature TS with AM led to a significant decrease of ATP and CrP and an increase of ADP compared with their levels before dilution, although immature TS were not motile. Energy dependency of TS maturation in sturgeon was confirmed, which suggests that mitochondrial oxidative phosphorylation is needed for maturation of sturgeon TS. PMID:26641041

  1. Inhibitory effect of caffeic acid on ADP-induced thrombus formation and platelet activation involves mitogen-activated protein kinases

    PubMed Central

    Lu, Yu; Li, Quan; Liu, Yu-Ying; Sun, Kai; Fan, Jing-Yu; Wang, Chuan-She; Han, Jing-Yan

    2015-01-01

    Caffeic acid (CA), one of the active constituents of Radix Salvia miltiorrhizae, exhibits antioxidant and anti-inflammatory activities. However, few studies have assessed the ability of CA to inhibit platelet mediated thrombus generation in vivo. In this study, we investigated the antithrombotic effect of CA in mouse cerebral arterioles and venules using intravital microscopy. The antiplatelet activity of CA in ADP stimulated mouse platelets in vitro was also examined in attempt to explore the underlying mechanism. Our results demonstrated that CA (1.25–5 mg/kg) significantly inhibited thrombus formation in vivo. In vitro, CA (25–100 μM) inhibited ADP-induced platelet aggregation, P-selectin expression, ATP release, Ca2+ mobilization, and integrin αIIbβ3 activation. Additionally, CA attenuated p38, ERK, and JNK activation, and enhanced cAMP levels. Taken together, these data provide evidence for the inhibition of CA on platelet-mediated thrombosis in vivo, which is, at least partly, mediated by interference in phosphorylation of ERK, p38, and JNK leading to elevation of cAMP and down-regulation of P-selectin expression and αIIbβ3 activation. These results suggest that CA may have potential for the treatment of aberrant platelet activation-related diseases. PMID:26345207

  2. Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of /sup 125/I-fibrinogen to rat platelets

    SciTech Connect

    McGregor, L.; Toor, B.; McGregor, J.L.; Renaud, S.; Clemetson, K.J.

    1984-03-01

    The aggregation to ADP and the binding of /sup 125/I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane.

  3. Adenosine and Ischemic Preconditioning

    PubMed Central

    Liang, Bruce T.; Swierkosz, Tomasz A.; Herrmann, Howard C.; Kimmel, Stephen; Jacobson, Kenneth A.

    2012-01-01

    Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent cardioprotective effects in the heart. Although these observations on adenosine have been known for a long time, how adenosine acts to achieve its anti-ischemic effect remains incompletely understood. However, recent advances on the chemistry and pharmacology of adenosine receptor ligands have provided important and novel information on the function of adenosine receptor subtypes in the cardiovascular system. The development of model systems for the cardiac actions of adenosine has yielded important insights into its mechanism of action and have begun to elucidate the sequence of signalling events from receptor activation to the actual exertion of its cardioprotective effect. The present review will focus on the adenosine receptors that mediate the potent anti-ischemic effect of adenosine, new ligands at the receptors, potential molecular signalling mechanisms downstream of the receptor, mediators for cardioprotection, and possible clinical applications in cardiovascular disorders. PMID:10607860

  4. Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS).

    PubMed

    Kim, Yun-Gon; Park, Hyung-Yeon; Yoo, Dongwon; Sung, Changmin; Song, Eunjung; Lee, Jae-Hun; Choi, Yun-Hui; Kim, Yong-Hyun; Lee, Chang-Soo; Park, Kyungmoon; Kim, Byung-Gee; Yang, Yung-Hun

    2012-04-15

    Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases. PMID:22459405

  5. Relationship between ADP-induced platelet-fibrin clot strength and anti-platelet responsiveness in ticagrelor treated ACS patients

    PubMed Central

    Li, Dan-Dan; Wang, Xu-Yun; Xi, Shao-Zhi; Liu, Jia; Qin, Liu-An; Jing, Jing; Yin, Tong; Chen, Yun-Dai

    2016-01-01

    Background Ticagrelor provides enhanced antiplatelet efficacy but increased risk of bleeding and dyspnea. This study aimed to display the relationship between ADP-induced platelet-fibrin clot strength (MAADP) and clinical outcomes in acute coronary syndrome (ACS) patients treated by ticagrelor. Methods Consecutive Chinese-Han patients with ACS who received maintenance dose of ticagrelor on top of aspirin were recruited. After 5-day ticagrelor maintenance treatment, MAADP measured by thrombelastography (TEG) were recorded for the evaluation of ticagrelor anti-platelet reactivity. Pre-specified cutoffs of MAADP > 47 mm for high on-treatment platelet reactivity (HTPR) and MAADP < 31 mm for low on-treatment platelet reactivity (LTPR) were applied for evaluation. The occurrences of primary ischemic cardiovascular events (including a composite of cardiac death, non-fatal myocardial infarction and stroke), the Thrombolysis in Myocardial Infarction (TIMI) defined bleeding events, and ticagrelor related dyspnea were recorded after a follow-up of three months. Results Overall, 176 ACS patients (Male: 79.55%, Age: 59.91 ± 10.54 years) under ticagrelor maintenance treatment were recruited. The value of MAADP ranged from 4.80% to 72.90% (21.27% ± 12.07% on average), with the distribution higher skewed towards the lower values. Using the pre-specific cutoffs for HTPR and LTPR, seven patients (3.98%) were identified as HTPR and 144 patients (81.82%) as LTPR. After a follow-up of three months in 172 patients, major cardiovascular events occurred in no patient, but TIMI bleeding events in 81 (47.09%) with major bleedings in three patients. All patients with major bleedings were classified as LTPR. Ticagrelor related dyspnea occurred in 31 (18.02%) patients, with 30 (21.28%) classified as LTPR and no one as HTPR (P = 0.02). Conclusions In ticagrelor treated ACS patients, MAADP measured by TEG might be valuable for the prediction of major bleeding and ticagrelor related dyspnea

  6. Nucleolin Down-Regulation Is Involved in ADP-Induced Cell Cycle Arrest in S Phase and Cell Apoptosis in Vascular Endothelial Cells

    PubMed Central

    Wang, Wenmeng; Luo, Junqing; Xiang, Fang; Liu, Xueting; Jiang, Manli; Liao, Lingjuan; Hu, Jinyue

    2014-01-01

    High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin. PMID:25290311

  7. Nucleolin down-regulation is involved in ADP-induced cell cycle arrest in S phase and cell apoptosis in vascular endothelial cells.

    PubMed

    Wang, Wenmeng; Luo, Junqing; Xiang, Fang; Liu, Xueting; Jiang, Manli; Liao, Lingjuan; Hu, Jinyue

    2014-01-01

    High concentration of extracellular ADP has been reported to induce cell apoptosis, but the molecular mechanisms remain not fully elucidated. In this study, we found by serendipity that ADP treatment of human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) down-regulated the protein level of nucleolin in a dose- and time-dependent manner. ADP treatment did not decrease the transcript level of nucloelin, suggesting that ADP might induce nucleolin protein degradation. HUVEC and HAEC expressed ADP receptor P2Y13 receptor, but did not express P2Y1 or P2Y12 receptors. However, P2Y1, 12, 13 receptor antagonists MRS2179, PSB0739, MRS2211 did not inhibit ADP-induced down-regulation of nucleolin. Moreover, MRS2211 itself down-regulated nucleolin protein level. In addition, 2-MeSADP, an agonist for P2Y1, 12 and 13 receptors, did not down-regulate nucleolin protein. These results suggested that ADP-induced nucleolin down-regulation was not due to the activation of P2Y1, 12, or 13 receptors. We also found that ADP treatment induced cell cycle arrest in S phase, cell apoptosis and cell proliferation inhibition via nucleolin down-regulation. The over-expression of nucleolin by gene transfer partly reversed ADP-induced cell cycle arrest, cell apoptosis and cell proliferation inhibition. Furthermore, ADP sensitized HUVEC to cisplatin-induced cell death by the down-regulation of Bcl-2 expression. Taken together, we found, for the first time to our knowledge, a novel mechanism by which ADP regulates cell proliferation by induction of cell cycle arrest and cell apoptosis via targeting nucelolin. PMID:25290311

  8. 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.

  9. Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules 1

    PubMed Central

    Nelson, Oliver E.; Chourey, Prem S.; Chang, Ming Tu

    1978-01-01

    Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent Km (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10−5 M, which is compared to 3 × 10−3 M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent Km estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction. PMID:16660522

  10. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  11. Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

    PubMed Central

    Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

    2012-01-01

    SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

  12. Relaxation of isolated taenia coli of guinea-pig by enantiomers of 2-azido analogues of adenosine and adenine nucleotides.

    PubMed Central

    Cusack, N. J.; Planker, M.

    1979-01-01

    1 2-Azido photoaffinity analogues of adenosine 5'triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine have been synthesized and tested on guinea-pig taenia coli. 2 2-Azido-ATP and 2-azido-ADP were approximately 20 times more potent than ATP as relaxants of taenia coli, and required prolonged washout times before recovery of the muscle. 3 2-Azido-AMP and 2-azidoadenosine were 2 to 12 times more potent than ATP, but took much longer (up to 100 s) to reach maximal relaxation. This behaviour is different from that of AMP and adenosine which were much less potent than ATP. 4 L-Enantiomers of adenosine and adenine nucleotides were also tested. L-ATP and L-ADP were 3 to 6 times less potent than ATP and ADP, and L-AMP and L-adenosine were inactive. 2-Azido-L-ATP and 2-azido-L-ADP were approximately 120 times less potent than 2-Azido-ATP and 6 times less potent than ATP as relaxants of taenia coli. 2-Azido-L-AMP and 2-azidio-L-adenosine were almost inactive. 5 2-Azido derivatives are photolysed by u.v. irradiation to reactive intermediates. 2-Azido-ATP and 2-azidoadenosine might be suitable photoaffinity ligands for labelling putative P2 and P1 purine receptors respectively. 2-Azido-L-ATP and 2-azido-L-adenosine could be useful controls for nonspecific labelling. PMID:497519

  13. Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N6-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  14. Fluorometric determination of adenosine nucleotide derivatives as measures of the microfouling, detrital, and sedimentary microbial biomass and physiological status.

    PubMed

    Davis, W M; White, D C

    1980-09-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

  15. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    SciTech Connect

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  16. Structure and function of uridine diphosphate glucuronosyltransferases.

    PubMed

    Meech, R; Mackenzie, P I

    1997-12-01

    1. The uridine diphosphate (UDP)-glucuronosyltransferases (UGT) are a family of enzymes that catalyse the covalent addition of glucuronic acid to a wide range of lipophilic chemicals. They play a major role in the detoxification of many exogenous and endogenous compounds by generating products that are more polar and, thus, more readily excreted in bile or urine. 2. Inherited deficiencies in UGT forms are deleterious, as exemplified by the debilitating effects of hyperbilirubinaemia and neurotoxicity in subjects with mutations in the enzyme that converts bilirubin to its more polar glucuronide. 3. The UGT protein can be conceptually divided into two domains with the amino-terminal half of the protein demonstrating greater sequence divergence between isoforms. This region apparently determines aglycone specificity. The aglycone binding site is presumed to be a 'loose' fit, as many structurally diverse substrates can be bound by the same UGT isoform. The carboxyl-terminal half, which is more conserved in sequence between different isoforms, is believed to contain a binding site for the cosubstrate UDP glucuronic acid (UDPGA). 4. Uridine diphosphate glucuronosyltransferase is localized to the endoplasmic reticulum (ER) and spans the membrane with a type I topology. The putative transmembrane domain is located near the carboxyl terminus of the protein such that only a small portion of the protein resides in the cytosol. This cytosolic tail is believed to contain an ER-targeting signal. The major portion of the protein is located in the ER lumen, including the proposed substrate-binding domains and the catalytic site. 5. The microsomal membrane impedes the access of UDPGA to the active site, resulting in latency of UGT activity in intact ER-derived microsomes. Active transport of UDPGA is believed to occur in hepatocytes, but the transport system has not been fully characterized. Uridine diphosphate glucuronosyltransferase activity is also highly lipid dependent and the

  17. Adenosine and Bone Metabolism

    PubMed Central

    Mediero, Aránzazu; Cronstein, Bruce N.

    2013-01-01

    Bone is a dynamic organ that undergoes continuous remodeling whilst maintaining a balance between bone formation and resorption. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, the cells that resorb bone, act in concert to maintain bone homeostasis. In recent years, there has been increasing appreciation of purinergic regulation of bone metabolism. Adenosine, released locally, mediates its physiologic and pharmacologic actions via interactions with G-protein coupled receptors and recent work has indicated that these receptors are involved in the regulation of osteoclast differentiation and function, as well as osteoblast differentiation and bone formation. Moreover, adenosine receptors also regulate chondrocyte and cartilage homeostasis. These recent findings underscore the potential therapeutic importance of adenosine receptors in regulating bone physiology and pathology. PMID:23499155

  18. Adenosine receptor interactions and anxiolytics.

    PubMed

    Bruns, R F; Katims, J J; Annau, Z; Snyder, S H; Daly, J W

    1983-12-01

    [3H]-N6-cyclohexyladenosine and [3H]-1,3-diethyl-8-phenylxanthine label the A1 subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A1 adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N6-(phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and N6-(L-phenylisopropyl)adenosine cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately. Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine. PMID:6199685

  19. Adenosine in fibrosis

    PubMed Central

    Chan, Edwin S. L.

    2011-01-01

    Adenosine is an endogenous autocoid that regulates a multitude of bodily functions. Its anti-inflammatory actions are well known to rheumatologists since it mediates many of the anti-inflammatory effects of a number of antirheumatic drugs such as methotrexate. However, inflammatory and tissue regenerative responses are intricately linked, with wound healing being a prime example. It has only recently been appreciated that adenosine has a key role in tissue regenerative and fibrotic processes. An understanding of these processes may shed new light on potential therapeutic options in diseases such as scleroderma where tissue fibrosis features prominently. PMID:19949965

  20. β-Arrestin 2-dependent activation of ERK1/2 is required for ADP-induced paxillin phosphorylation at Ser83 and microglia chemotaxis

    PubMed Central

    Lee, Sang-Hyun; Hollingsworth, Ryan; Kwon, Hyeok-Yil; Lee, Narae; Chung, Chang Y.

    2014-01-01

    Microglia play crucial roles in increased inflammation in the CNS upon brain injuries and diseases. Extracellular ADP has been reported to induce microglia chemotaxis and membrane ruffle formation through P2Y12 receptor. In this study, we examined the role of ERK1/2 activation in ADP-induced microglia chemotaxis. ADP stimulation increases the phosphorylation of ERK1/2 and paxillin phosphorylation at Tyr31 and Ser83. Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser83 and the retraction of membrane ruffles, causing inefficient chemotaxis. Close examination of dynamics of focal adhesion formation with GFP-paxillin revealed that the disassembly of focal adhesions in U0126-treated cells was significantly impaired. Depletion of β-Arr2 with shRNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser83, indicating that β-Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and β-Arr2 were translocated and co-localized at focal contacts in the newly forming lamellipodia. Examination of kinetics and rate constant of paxillin formation and disassembly revealed that the phosphorylation of paxillin at Tyr31 by c-Src appears to be involved in adhesion formation upon ADP stimulation while Ser83 required for adhesion disassembly. PMID:22638989

  1. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  2. Role of Adenosine Signaling on Pentylenetetrazole-Induced Seizures in Zebrafish

    PubMed Central

    Siebel, Anna Maria; Menezes, Fabiano Peres; Capiotti, Katiucia Marques; Kist, Luiza Wilges; Schaefer, Isabel da Costa; Frantz, Juliana Zanetti; Bogo, Maurício Reis; Da Silva, Rosane Souza

    2015-01-01

    Abstract Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5′nucleotidase inhibitor adenosine 5′-(α,β-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5′-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish. PMID:25560904

  3. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

    PubMed Central

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  4. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine.

    PubMed

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

  5. Construction of Functional Monomeric Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase.

    PubMed

    Neti, Syam Sundar; Eckert, Debra M; Poulter, C Dale

    2016-08-01

    Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the isoprenoid biosynthetic pathway. The enzyme from Streptomyces pneumoniae (spIDI-2) is a homotetramer in solution with behavior, including a substantial increase in the rate of FMN reduction by NADPH in the presence of IPP, suggesting that substrate binding at one subunit alters the kinetic and binding properties of another. We now report the construction of catalytically active monomeric spIDI-2. The monomeric enzyme contains a single-point mutation (N37A) and a six-residue C-terminal deletion that preserves the secondary structure of the subunits in the wild-type (wt) homotetramer. UV-vis spectra of the enzyme-bound flavin mononucleotide (FMN) cofactor in FMNox, FMNred, and FMNred·IPP/DMAPP states are the same for monomeric and wt homotetrameric spIDI-2. The mutations in monomeric IDI-2 lower the melting temperature of the protein by 20 °C and reduce the binding affinities of FMN and IDI by 40-fold but have a minimal effect on kcat. Stopped-flow kinetic studies of monomeric spIDI-2 showed that the rate of reduction of FMN by NADH (k = 1.64 × 10(-3) s(-1)) is substantially faster when IPP is added to the monomeric enzyme (k = 0.57 s(-1)), similar to behavior seen for wt-spIDI-2. Our results indicate that cooperative interactions among subunits in the wt homotetramer are not responsible for the increased rate of reduction of spIDI-2·FMN by NADH, and two possible scenarios for the enhancement are suggested. PMID:27379573

  6. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    SciTech Connect

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-02-26

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  7. Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

    SciTech Connect

    Sanchez, Elda E.; Galan, Jacob A.; Russell, William K.; Soto, Julio G.; Russell, David H.; Perez, John C. . E-mail: kfjcp00@tamuk.edu

    2006-04-01

    Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC{sub 5}s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.

  8. Adenosine kinase inhibitors attenuate opiate withdrawal via adenosine receptor activation.

    PubMed

    Kaplan, G B; Coyle, T S

    1998-11-27

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. This study examines the effects of indirect activation of adenosine receptors, via treatment with adenosine kinase inhibitors, on the expression of opiate withdrawal in mice. Mice receive chronic morphine treatment via implantation of subcutaneous morphine pellets (75 mg) for 72 h. Mice then receive parenteral treatment with adenosine kinase inhibitors, either 5'-amino-5'-deoxyadenosine (2, 5, 20, 40 mg/kg, intraperitoneal or i.p.) or iodotubericidin (1, 2, 5 mg/kg, i.p.), followed by naloxone injection and opiate withdrawal signs are measured over 20 min. Both adenosine kinase inhibitors significantly reduce the following opiate withdrawal signs in a dose-dependent manner compared to vehicle: withdrawal jumps, teeth chattering, forepaw tremors, and forepaw treads. Additionally, 5'-amino-5'-deoxyadenosine significantly reduces withdrawal-induced diarrhea and weight loss. Effects of 5'-amino-5'-deoxyadenosine (40 mg/kg) on opiate withdrawal signs appear to be mediated via adenosine receptor activation as they are reversed by pretreatment by adenosine receptor antagonist caffeine (20 mg, i.p.) but not by selective phosphodiesterase inhibitor Ro 20-1724 (10 mg/kg, i.p.). Adenosine receptor activation via adenosine kinase inhibitor treatment attenuates opiate withdrawal and these agents may be generally useful in the treatment of drug withdrawal syndromes. PMID:9865523

  9. Structure-Based Design, Synthesis, and Evaluation of 2'-(2-Hydroxyethyl)-2'-deoxyadenosine and the 5'-Diphosphate Derivative as Ribonucleotide Reductase Inhibitors

    SciTech Connect

    Sun, D.; Xu, H.; Wijerathna, S.R.; Dealwis, C.; Lee, R.E.

    2010-08-27

    Analysis of the recently solved X-ray crystal structures of Saccharomyces cerevisiae ribonucleotide reductase I (ScRnr1) in complex with effectors and substrates led to the discovery of a conserved water molecule located at the active site that interacted with the 2'-hydroxy group of the nucleoside ribose. In this study 2'-(2-hydroxyethyl)-2'-deoxyadenosine 1 and the 5'-diphosphate derivative 2 were designed and synthesized to see if the conserved water molecule could be displaced by a hydroxymethylene group, to generate novel RNR inhibitors as potential antitumor agents. Herein we report the synthesis of analogues 1 and 2, and the co-crystal structure of adenosine diphosphate analogue 2 bound to ScRnr1, which shows the conserved water molecule is displaced as hypothesized.

  10. Adenosine receptor desensitization and trafficking.

    PubMed

    Mundell, Stuart; Kelly, Eamonn

    2011-05-01

    As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. PMID:20550943

  11. Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates.

    PubMed

    Nagaki, Masahiko; Nakada, Minori; Musashi, Tohru; Kawakami, Jun; Ohya, Norimasa; Kurihara, Masayo; Maki, Yuji; Nishino, Tokuzo; Koyama, Tanetoshi

    2007-07-01

    To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained. PMID:17617711

  12. Properties of ribulose diphosphate carboxylase immobilized on porous glass

    NASA Technical Reports Server (NTRS)

    Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

    1974-01-01

    Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

  13. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

    ... Health Conditions adenosine deaminase 2 deficiency adenosine deaminase 2 deficiency Enable Javascript to view the expand/collapse ... PDF Open All Close All Description Adenosine deaminase 2 (ADA2) deficiency is a disorder characterized by abnormal ...

  14. Neurochemical measurement of adenosine in discrete brain regions of five strains of inbred mice.

    PubMed

    Pani, Amar K; Jiao, Yun; Sample, Kenneth J; Smeyne, Richard J

    2014-01-01

    Adenosine (ADO), a non-classical neurotransmitter and neuromodulator, and its metabolites adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP), have been shown to play an important role in a number of biochemical processes. Although their signaling is well described, it has been difficult to directly, accurately and simultaneously quantitate these purines in tissue or fluids. Here, we describe a novel method for measuring adenosine (ADO) and its metabolites using high performance liquid chromatography with electrochemical detection (HPLC-ECD). Using this chromatographic technique, we examined baseline levels of ADO and ATP, ADP and AMP in 6 different brain regions of the C57BL/6J mouse: stratum, cortex, hippocampus, olfactory bulb, substantia nigra and cerebellum and compared ADO levels in 5 different strains of mice (C57BL/6J, Swiss-Webster, FVB/NJ, 129P/J, and BALB/c). These studies demonstrate that baseline levels of purines vary significantly among the brain regions as well as between different mouse strains. These dissimilarities in purine concentrations may explain the variable phenotypes among background strains described in neurological disease models. PMID:24642754

  15. Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase.

    PubMed

    Janczak, Matthew Walter; Poulter, C Dale

    2016-04-19

    Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) converts isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), the two fundamental building blocks of isoprenoid molecules. IDI-2 is found in many species of bacteria and is a potential antibacterial target since this isoform is non-homologous to the type 1 enzyme in Homo sapiens. IDI-2 requires a reduced flavin mononucleotide to form the catalytically active ternary complex, IDI-2·FMNH2·IPP. For IDI-2 from the pathogenic bacterium Streptococcus pneumoniae, the flavin can be treated kinetically as a dissociable cosubstrate in incubations with IPP and excess NADH. Under these conditions, the enzyme follows a modified sequential ordered mechanism where FMN adds before IPP. Interestingly, the enzyme shows sigmoidal behavior when incubated with IPP and NADH with varied concentrations of FMN in aerobic conditions. In contrast, sigmoidal behavior is not seen in incubations under anaerobic conditions where FMN is reduced to FMNH2 before the reaction is initiated by addition of IPP. Stopped-flow experiments revealed that FMN, whether bound to IDI-2 or without enzyme in solution, is slowly reduced in a pseudo-first-order reaction upon addition of excess NADH (kred(FMN) = 5.7 × 10(-3) s(-1) and kred(IDI-2·FMN) = 2.8 × 10(-3) s(-1)), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2·FMN, and IPP (kred(IDI-2·FMN·IPP) = 8.9 s(-1)). Similar experiments with dithionite as the reductant gave kred(FMN) = 221 s(-1) and kred(IDI-2·FMN) = 411 s(-1). Dithionite reduction of FMN in the IDI-2·FMN and IPP mixture was biphasic with kred(IDI-2·FMN·IPP (fast)) = 326 s(-1) and kred(IDI-2·FMN·IPP (slow)) = 6.9 s(-1) The pseudo-first-order rate constant for the slow component was similar to those for NADH reduction of the flavin in the IDI-2·FMN and IPP mixture and may reflect a rate-limiting conformational change in the enzyme. PMID:27003727

  16. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    PubMed

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

  17. [Adenosine and its role in physiology].

    PubMed

    Novotný, J

    2015-01-01

    Adenosine is not just a major component of adenine nucleotides and ribonucleic acids, but also has its own signaling functions. ExtraceIlular level of adenosine in an organism is strictly maintained through the balance between its formation, degradation and transport. Adenosine is formed by enzymatic degradation of adenosine triphosphate and eliminated by phosphorylation to adenosine monophosphate or by deamination to inosine. Transport of adenosine across the cell membrane is ensured by equilibrative and concentrative nucleoside transporters. All these processes participate in maintenance of adenosine level under normal conditions, but a balanced equilibrium can be disrupted in some pathophysiological situations. Extracellular adenosine as a signaling molecule binds to adenosine receptors, which may trigger via their cognate trimeric G proteins different signaling pathways. In this way, adenosine regulates energy homeostasis and affects the function of various organs. Targeted pharmacological manipulations of specific adenosine receptor subtypes or enzymes involved in its metabolism can potentially be used for therapeutic purposes. PMID:26738245

  18. Presence of phosphorylated O-ribosyl-adenosine in T-psi-stem of yeast methionine initiator tRNA.

    PubMed Central

    Desgrès, J; Keith, G; Kuo, K C; Gehrke, C W

    1989-01-01

    We report in this paper on isolation and characterization of two unknown nucleosides G* and [A*] located in the T-psi-stem of yeast methionine initiator tRNA, using the combined means of HPLC protocols, real time UV-absorption spectrum, and post-run mass spectrometry by electron impact or fast atom bombardment. The G* nucleoside in position 65 was identified as unmodified guanosine. The structure of the unknown [A*] in position 64 was characterized as an isomeric form of O-ribosyl-adenosine by comparison of its chromatographic, UV-spectral and mass spectrometric properties with those of authentic O-alpha-ribofuranosyl-(1"----2')-adenosine isolated from biosynthetic poly(adenosine diphosphate ribose). Our studies also brought evidence for the presence of a phosphorylmonoester group located on this new modified nucleoside [A*], when isolated by ion exchange chromatography from enzymic hydrolysis of yeast initiator tRNAMet without phosphatase treatment. PMID:2646591

  19. Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

    PubMed

    Lopez-Zavala, Alonso A; Sotelo-Mundo, Rogerio R; Hernandez-Flores, Jose M; Lugo-Sanchez, Maria E; Sugich-Miranda, Rocio; Garcia-Orozco, Karina D

    2016-06-01

    Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme. PMID:27072556

  20. Taxodione and arenarone inhibit farnesyl diphosphate synthase by binding to the isopentenyl diphosphate site.

    PubMed

    Liu, Yi-Liang; Lindert, Steffen; Zhu, Wei; Wang, Ke; McCammon, J Andrew; Oldfield, Eric

    2014-06-24

    We used in silico methods to screen a library of 1,013 compounds for possible binding to the allosteric site in farnesyl diphosphate synthase (FPPS). Two of the 50 predicted hits had activity against either human FPPS (HsFPPS) or Trypanosoma brucei FPPS (TbFPPS), the most active being the quinone methide celastrol (IC50 versus TbFPPS ∼ 20 µM). Two rounds of similarity searching and activity testing then resulted in three leads that were active against HsFPPS with IC50 values in the range of ∼ 1-3 µM (as compared with ∼ 0.5 µM for the bisphosphonate inhibitor, zoledronate). The three leads were the quinone methides taxodone and taxodione and the quinone arenarone, compounds with known antibacterial and/or antitumor activity. We then obtained X-ray crystal structures of HsFPPS with taxodione+zoledronate, arenarone+zoledronate, and taxodione alone. In the zoledronate-containing structures, taxodione and arenarone bound solely to the homoallylic (isopentenyl diphosphate, IPP) site, not to the allosteric site, whereas zoledronate bound via Mg(2+) to the same site as seen in other bisphosphonate-containing structures. In the taxodione-alone structure, one taxodione bound to the same site as seen in the taxodione+zoledronate structure, but the second located to a more surface-exposed site. In differential scanning calorimetry experiments, taxodione and arenarone broadened the native-to-unfolded thermal transition (Tm), quite different to the large increases in ΔTm seen with biphosphonate inhibitors. The results identify new classes of FPPS inhibitors, diterpenoids and sesquiterpenoids, that bind to the IPP site and may be of interest as anticancer and antiinfective drug leads. PMID:24927548

  1. Positive control of lac operon expression in vitro by guanosine 5'-diphosphate 3'-diphosphate.

    PubMed

    Primakoff, P; Artz, S W

    1979-04-01

    Maximal expression of the Escherichia coli lactose operon in a coupled in vitro transcription-translation system from a Salmonella typhimurium relA mutant was strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Without added ppGpp, at saturating 3',5'-cyclic AMP (cAMP) concentrations, synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was reproducibly only 5-7% of that which can be obtained with 0.5-0.8 mM ppGpp. Experiments in which transcription was uncoupled from translation indicated that this 14- to 20-fold stimulation by ppGpp occurred at the level of transcription. When coupled beta-galactosidase synthesis was primed with a template containing a well-characterized mutant lac promoter (lacP(r)L8UV5), the dependence on ppGpp was greatly reduced. This result provides an important experimental control previously unavailable for verifying the significance of ppGpp effects on gene regulation in vitro; it indicates that activation of lacP(+) expression by ppGpp is specifically an effect of increased transcription initiations. Furthermore, the large ppGpp stimulation of lacP(+) DNA enabled the level of expression of this template to approach that of lacP(r)L8UV5 DNA, an observation expected from results in vivo but not obtained with other transcription-translation systems in vitro. The importance of these results is considered with respect to previous ideas on the physiological role of ppGpp as a supercontrol molecule in bacterial regulation. PMID:109832

  2. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    PubMed

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon. PMID:27338660

  3. Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

    PubMed

    Sitthithaworn, W; Kojima, N; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Noji, M; Saito, K; Niwa, Y; Sankawa, U

    2001-02-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein. PMID:11217109

  4. Adenosine-Associated Delivery Systems

    PubMed Central

    Kazemzadeh-Narbat, Mehdi; Annabi, Nasim; Tamayol, Ali; Oklu, Rahmi; Ghanem, Amyl; Khademhosseini, Ali

    2016-01-01

    Adenosine is a naturally occurring purine nucleoside in every cell. Many critical treatments such as modulating irregular heartbeat (arrhythmias), regulation of central nervous system (CNS) activity, and inhibiting seizural episodes can be carried out using adenosine. Despite the significant potential therapeutic impact of adenosine and its derivatives, the severe side effects caused by their systemic administration have significantly limited their clinical use. In addition, due to adenosine’s extremely short half-life in human blood (less than 10 s), there is an unmet need for sustained delivery systems to enhance efficacy and reduce side effects. In this paper, various adenosine delivery techniques, including encapsulation into biodegradable polymers, cell-based delivery, implantable biomaterials, and mechanical-based delivery systems, are critically reviewed and the existing challenges are highlighted. PMID:26453156

  5. Characterization of ribulose diphosphate carboxylase and phosphoribulokinase from Thiobacillus thioparus and Thiobacillus neapolitanus.

    NASA Technical Reports Server (NTRS)

    Johnson, E. J.; Johnson, M. K.; Macelroy, R. D.

    1968-01-01

    Ribulose diphosphate carboxylase and phosphoribulokinase activity in chemosynthetic autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus, noting sedimentation and gel filtration characteristics

  6. Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

    NASA Technical Reports Server (NTRS)

    Mar, A.; Dworkin, J.; Oro, J.

    1987-01-01

    Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

  7. Purification of geranylgeranyl diphosphate synthase from bovine brain.

    PubMed

    Sagami, H; Morita, Y; Korenaga, T; Ogura, K

    1994-01-01

    Geranylgeranyl diphosphate (GGPP) synthase was purified to homogeneity from bovine brain in a one-step affinity column procedure. For the construction of the affinity column, a farnesyl diphosphate (FPP) analog, O-(6-amino-1-hexyl)-P-farnesylmethyl phosphonophosphate, was synthesized and linked to the spacer of the matrix of Affigel 10 via the amino group. The native enzyme appeared to be homooligomer (150-195 kDa) with a molecular mass of the monomer of 37.5 kDa. The pI for the enzyme was 6.2. The Km values for dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP) and FPP were estimated to be 33 microM, 0.80 microM and 0.74 microM, respectively. The Km value for isopentenyl diphosphate (IPP) in the presence of both IPP and FPP mixture was 2 microM. The ratio of the reaction velocity for formation of GGPP from DMAPP, GPP or FPP was 0.004:0.145:1. The intermediate FPP was formed in the reaction with GPP as an allylic primer. FPP synthase catalyzing the formation of FPP from DMAPP and IPP was also purified to homogeneity from the same organ by a similar affinity chromatography procedure using a GPP analog, O-(6-amino-1-hexyl)-P-geranylmethyl phosphonophosphate as a ligand. The enzyme was a homodimer with a monomeric molecular mass of 40.0 kDa. These results indicate that GGPP, a lipid precursor for the biosynthesis of a majority of prenylated proteins, is synthesized from DMAPP and IPP by the action of FPP synthase catalyzing the reactions C5-->C15 followed by the action of GGPP synthase catalyzing the reaction C15-->C20. PMID:7856400

  8. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

    PubMed Central

    López-Zavala, Alonso A.; Quintero-Reyes, Idania E.; Carrasco-Miranda, Jesús S.; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.

    2014-01-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012 ▶), J. Bioenerg. Biomembr. 44, 325–331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2′-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  9. Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates.

    PubMed

    López-Zavala, Alonso A; Quintero-Reyes, Idania E; Carrasco-Miranda, Jesús S; Stojanoff, Vivian; Weichsel, Andrzej; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R

    2014-09-01

    Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism. PMID:25195883

  10. Extracellular guanosine regulates extracellular adenosine levels

    PubMed Central

    Cheng, Dongmei; Jackson, Travis C.; Verrier, Jonathan D.; Gillespie, Delbert G.

    2013-01-01

    The aim of this investigation was to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels. Rat preglomerular vascular smooth muscle cells were incubated with adenosine, guanosine, or both. Guanosine (30 μmol/l) per se had little effect on extracellular adenosine levels. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) were 0.125 ± 0.020 μmol/l, indicating rapid disposition of extracellular adenosine. Extracellular adenosine levels 1 h after addition of adenosine (3 μmol/l) plus guanosine (30 μmol/l) were 1.173 ± 0.061 μmol/l, indicating slow disposition of extracellular adenosine. Cell injury increased extracellular levels of endogenous adenosine and guanosine, and the effects of cell injury on endogenous extracellular adenosine were modulated by altering the levels of endogenous extracellular guanosine with exogenous purine nucleoside phosphorylase (converts guanosine to guanine) or 8-aminoguanosine (inhibits purine nucleoside phosphorylase). Extracellular guanosine also slowed the disposition of extracellular adenosine in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts, and kidney epithelial cells and in human aortic and coronary artery vascular smooth muscle cells and coronary artery endothelial cells. The effects of guanosine on adenosine levels were not mimicked or attenuated by 5-iodotubericidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)-adenine (adenosine deaminase inhibitor), 5-aminoimidazole-4-carboxamide (guanine deaminase inhibitor), aristeromycin (S-adenosylhomocysteine hydrolase inhibitor), low sodium (inhibits concentrative nucleoside transporters), S-(4-nitrobenzyl)−6-thioinosine [inhibits equilibrative nucleoside transporter (ENT) type 1], zidovudine (inhibits ENT type 2), or acadesine (known modulator of adenosine levels). Guanosine also increases extracellular inosine, uridine, thymidine, and cytidine, yet decreases

  11. Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine.

    PubMed

    Valenti, D; Barile, M; Quagliariello, E; Passarella, S

    1999-02-12

    The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase. PMID:10050777

  12. Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate☆

    PubMed Central

    Karp, Frank; Zhao, Yuxin; Santhamma, Bindu; Assink, Bryce; Coates, Robert M.; Croteau, Rodney B.

    2007-01-01

    The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (−)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase. PMID:17949678

  13. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    MedlinePlus

    Skip to main content Your Guide to Understanding Genetic Conditions Enable Javascript for addthis links to activate. ... Conditions Genes Chromosomes & mtDNA Resources Help Me Understand Genetics Home Health Conditions adenosine monophosphate deaminase deficiency adenosine ...

  14. Impaired Erectile Function in CD73-deficient Mice with Reduced Endogenous Penile Adenosine Production

    PubMed Central

    Wen, Jiaming; Dai, Yingbo; Zhang, Yujin; Zhang, Weiru; Kellems, Rodney E.; Xia, Yang

    2012-01-01

    Introduction Adenosine has been implicated in normal and abnormal penile erection. However, a direct role of endogenous adenosine in erectile physiology and pathology has not been established. Aim To determine the functional role of endogenous adenosine production in erectile function. Methods CD73-deficient mice (CD73−/−) and age-matched wild-type (WT) mice were used. Some WT mice were treated with alpha, beta-methylene adenosine diphosphate (ADP) (APCP), a CD73-specific inhibitor. High-performance liquid chromatography was used to measure adenosine levels in mouse penile tissues. In vivo assessment of intracorporal pressure (ICP) normalized to mean arterial pressure (MAP) in response to electrical stimulation (ES) of the cavernous nerve was used. Main Outcome Measurement The main outcome measures of this study were the in vivo assessment of initiation and maintenance of penile erection in WT mice and mice with deficiency in CD73 (ecto-5′-nucleotidase), a key cell-surface enzyme to produce extracellular adenosine. Results Endogenous adenosine levels were elevated in the erected state induced by ES of cavernous nerve compared to the flaccid state in WT mice but not in CD73−/− mice. At cellular levels, we identified that CD73 was highly expressed in the neuronal, endothelial cells, and vascular smooth muscle cells in mouse penis. Functionally, we found that the ratio of ES-induced ICP to MAP in CD73−/− mice was reduced from 0.48 ± 0.03 to 0.33 ± 0.05 and ES-induced slope was reduced from 0.30 ± 0.13 mm Hg/s to 0.15 ± 0.05 mm Hg/s (both P < 0.05). The ratio of ES-induced ICP to MAP in APCP-treated WT mice was reduced from 0.49 ± 0.03 to 0.38 ± 0.06 and ES-induced slope was reduced from 0.29 ± 0.11 mm Hg/s to 0.19 ± 0.04 mm Hg/s (both P < 0.05). Conclusion Overall, our findings demonstrate that CD73-dependent production of endogenous adenosine plays a direct role in initiation and maintenance of penile erection. PMID:21595838

  15. Synthesis, Raman and Rietveld analysis of thorium diphosphate

    SciTech Connect

    Clavier, Nicolas Dacheux, Nicolas; Beaunier, Patricia

    2008-12-15

    We report the synthesis of thorium diphosphate ThP{sub 2}O{sub 7} and its study by Raman spectroscopy and Rietveld analysis. This compound has been found to be closely related to the zirconium diphosphate type, with space group Pa-3 and a=8.7601(3) A. No superstructure was observed. The metastability of ThP{sub 2}O{sub 7} appears to stem from the six-fold oxygen environment of Th{sup IV}, a unique case in the structural chemistry of this cation. - Grapical abstract: The cubic structure of ThP{sub 2}O{sub 7}, built of ThO{sub 6} octahedra and P{sub 2}O{sub 7} ditetrahedra.

  16. Adenosine Receptors and Membrane Microdomains

    PubMed Central

    Lasley, Robert D.

    2010-01-01

    Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors (GPCR). The four adenosine receptor subtypes – A1, A2a, A2b, A3 – exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of GPCR signaling at the level of protein-protein interactions as well as through signaling crosstalk. With respect to adenosine receptors the activation of one receptor subtype can have profound direct effects in one cell type, but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of GPCR signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling. PMID:20888790

  17. Xanthines as Adenosine Receptor Antagonists

    PubMed Central

    Jacobson, Kenneth A.

    2013-01-01

    The natural plant alkaloids caffeine and theophylline were the first adenosine receptor (AR) antagonists described in the literature. They exhibit micromolar affinities and are non-selective. A large number of derivatives and analogs have subsequently been synthesized and evaluated as AR antagonists. Very potent antagonists have thus been developed with selectivity for each of the four AR subtypes. PMID:20859796

  18. Regulation of adenosine levels during cerebral ischemia

    PubMed Central

    Chu, Stephanie; Xiong, Wei; Zhang, Dali; Soylu, Hanifi; Sun, Chao; Albensi, Benedict C; Parkinson, Fiona E

    2013-01-01

    Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events, and attenuates the excitotoxic neuronal injury. Adenosine is produced both intracellularly and extracellularly, and nucleoside transport proteins transfer adenosine across plasma membranes. Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption, cellular release of ATP, metabolism of extracellular ATP (and other adenine nucleotides), adenosine influx, adenosine efflux and adenosine metabolism. Recent studies have used genetically modified mice to investigate the relative contributions of intra- and extracellular pathways for adenosine formation. The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase. From these studies, we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures, but not in hippocampal slices or in vivo mice exposed to ischemic conditions. PMID:23064722

  19. Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5-Epi-Aristolochene Synthase

    PubMed Central

    Rising, Kathleen A.; Crenshaw, Charisse M.; Koo, Hyun Jo; Subramanian, Thangaiah; Chehade, Kareem A. H.; Starks, Courtney; Allen, Keith D.; Andres, Douglas A.; Spielmann, H. Peter; Noel, Joseph P.; Chappell, Joe

    2015-01-01

    As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids. PMID:25897591

  20. Determination of kinetics and crystal structure of a novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase from Streptococcus pneumoniae

    PubMed Central

    de Ruyck, Jerome; Janczak, Matthew W.; Neti, Syam Sundar; Rothman, Steven C.; Schubert, Heidi L.; Cornish, Rita M.; Matagne, Andre; Wouters, Johan; Poulter, C. Dale

    2014-01-01

    Isopentenyl diphosphate dimethylallyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein and is found in eukaryotes, while the type-2 isoform (IDI-2) is a flavoenzyme found in bacteria and completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in E. coli. Steady state kinetic studies of the enzyme indicated that FMNH2 (KM= 0.3 μM) bound before isopentenyl diphosphate (KM= 40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 Å resolution was obtained for the holo-enzyme, in the closed conformation with reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against closely sequence and structure related pathogens such as E. faecalis or S. aureus. PMID:24910111

  1. Adenine nucleoside diphosphates block adaptation of mechanoelectrical transduction in hair cells.

    PubMed

    Gillespie, P G; Hudspeth, A J

    1993-04-01

    By adapting to sustained stimuli, hair cells in the internal ear retain their sensitivity to minute transient displacements. Because one model for adaptation asserts that this process is mediated by a myosin isozyme, we reasoned that we should be able to arrest adaptation by interfering with myosin's ATPase cycle though introduction of ADP into hair cells. During tight-seal, whole-cell recordings of transduction currents in cells isolated from bullfrog (Rana catesbeiana) sacculus, dialysis with 5-25 mM ADP gave variable results. In half of the cells examined, the rate of adaptation remained unchanged or even increased; adaptation was blocked in the remaining cells. Because we suspected that the variable effect of ADP resulted from the conversion of ADP to ATP by adenylate kinase, we employed the ADP analog adenosine 5'-[beta-thio]diphosphate (ADP[beta S]), which is not a substrate for adenylate kinase. Adaptation consistently disappeared in the presence of 1-10 mM ADP[beta S]; in addition, the transduction channels' open probability at rest grew from approximately 0.1 to 0.8 or more. Both effects could be reversed by 2 mM ATP. When used in conjunction with the adenylate kinase inhibitor P1,P5-bis(5'-adenosyl) pentaphosphate (Ap5A), ADP had effects similar to those of ADP[beta S]. These results suggest that adaptation by hair cells involves adenine nucleotides, and they lend support to the hypothesis that the adaptation process is powered by a myosin motor. PMID:8464880

  2. Acid-Base and Metal Ion-Coordinating Properties of Pyrimidine-Nucleoside 5'-Diphosphates (CDP, UDP, dTDP) and of Several Simple Diphosphate Monoesters. Establishment of Relations between Complex Stability and Diphosphate Basicity.

    PubMed

    Sajadi, S. Ali A.; Song, Bin; Gregán, Fridrich; Sigel, Helmut

    1999-02-01

    The stability constants of the 1:1 complexes formed between Mg(2+), Ca(2+), Sr(2+), Ba(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), or Cd(2+) and the pyrimidine-nucleoside 5'-diphosphates CDP(3)(-), UDP(3)(-), and dTDP(3)(-) (= NDP(3)(-)) were determined by potentiometric pH titration in aqueous solution (I = 0.1 M, NaNO(3); 25 degrees C). For comparison, the same values were measured for the corresponding complexes with the simple diphosphate monoesters (R-DP(3)(-)) phenyl diphosphate, methyl diphosphate, and n-butyl diphosphate. The acidity constants for H(3)(CDP)(+/-), H(2)(UDP)(-), H(2)(dTDP)(-), and H(2)(R-DP)(-) were measured also via potentiometric pH titration and various comparisons with related constants are made. By plotting log versus for the complexes of all six diphosphates mentioned and by a careful evaluation of the deviation of the various data pairs from the straight-line correlations, the expectation is confirmed that in the M(UDP)(-) and M(dTDP)(-) complexes the metal ion is only diphosphate-coordinated. The straight-line equations, which result from the mentioned correlations, together with the pK(a) value of a given monoprotonated diphosphate monoester allow now to predict the stability of the corresponding M(R-DP)(-) complexes. In this way, the experimentally determined stability constants for the M(CDP)(-) complexes are evaluated and it is concluded that the pyridine-like N3 of the cytosine residue does not participate in complex formation; i.e., the stability of the M(CDP)(-) complexes is also solely determined by the coordination tendency of the diphosphate residue. In all the monoprotonated M(H;NDP) and M(H;R-DP) complexes both, H(+) and M(2+), are bound at the diphosphate group. Only the Cu(H;CDP) complex exists in aqueous solution in the form of three different isomers: about 15% of the species have Cu(2+) and H(+) at the diphosphate residue, in about 13% Cu(2+) is bound at N3 and H(+) at the terminal beta-phosphate group, and the

  3. Adenosine/guanosine-3',5'-bis-phosphates as biocompatible and selective Zn2+-ion chelators. Characterization and comparison with adenosine/guanosine-5'-di-phosphate.

    PubMed

    Sayer, Alon Haim; Blum, Eliav; Major, Dan Thomas; Vardi-Kilshtain, Alexandra; Levi Hevroni, Bosmat; Fischer, Bilha

    2015-04-28

    Although involved in various physiological functions, nucleoside bis-phosphate analogues and their metal-ion complexes have been scarcely studied. Hence, here, we explored the solution conformation of 2′-deoxyadenosine- and 2′-deoxyguanosine-3′,5′-bisphosphates, 3 and 4, d(pNp), as well as their Zn(2+)/Mg(2+) binding sites and binding-modes (i.e. inner- vs. outer-sphere coordination), acidity constants, stability constants of their Zn(2+)/Mg(2+) complexes, and their species distribution. Analogues 3 and 4, in solution, adopted a predominant Southern ribose conformer (ca. 84%), gg conformation around C4'-C5' and C5'-O5' bonds, and glycosidic angle in the anti-region (213-270°). (1)H- and (31)P-NMR experiments indicated that Zn(2+)/Mg(2+) ions coordinated to P5' and P3' groups of 3 and 4 but not to N7 nitrogen atom. Analogues 3 and 4 formed ca. 100-fold more stable complexes with Zn(2+)vs. Mg(2+)-ions. Complexes of 3 and 4 with Mg(2+) at physiological pH were formed in minute amounts (11% and 8%, respectively) vs. Zn(2+) complexes (46% and 44%). Stability constants of Zn(2+)/Mg(2+) complexes of analogues 3 and 4 (log KML(M) = 4.65-4.75/2.63-2.79, respectively) were similar to those of the corresponding complexes of ADP and GDP (log KML(M) = 4.72-5.10/2.95-3.16, respectively). Based on the above findings, we hypothesized that the unexpectedly low log K values of Zn(2+)-d(pNp) as compared to Zn(2+)-NDP complexes, are possibly due to formation of outer-sphere coordination in the Zn(2+)-d(pNp) complex vs. inner-sphere in the NDP-Zn(2+) complex, in addition to loss of chelation to N7 nitrogen atom in Zn(2+)-d(pNp). Indeed, explicit solvent molecular dynamics simulations of 1 and 3 for 100 ns supported this hypothesis. PMID:25797179

  4. Kinetic studies of the reverse reaction catalysed by adenosine triphosphate–creatine phosphotransferase. The inhibition by magnesium ions and adenosine diphosphate

    PubMed Central

    Morrison, J. F.; O'Sullivan, W. J.

    1965-01-01

    1. Kinetic investigations of the reaction catalysed by ATP–creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg2+ at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion–nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg2+ is a non-competitive inhibitor of the enzyme with respect to both MgADP− and phosphocreatine. 4. ADP3− is a competitive inhibitor of the enzyme with respect to MgADP− and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants. PMID:14342234

  5. Unexpected reactivity of 2-fluorolinalyl diphosphate in the active site of crystalline 2-methylisoborneol synthase

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2013-01-01

    The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in its unliganded state and in complex with 2 Mg2+ ions and cis-2-fluorogeranyl diphosphate at 1.85 Å and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally-occurring, non-canonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization-isomerization-ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only 2 Mg2+ ions. Assays in solution indicate that MIBS catalyzes the generation of (1R)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1R)-(+)-camphor. PMID:23844678

  6. Fluorescent Ligands for Adenosine Receptors

    PubMed Central

    Kozma, Eszter; Jayasekara, P Suresh; Squarcialupi, Lucia; Paoletta, Silvia; Moro, Stefano; Federico, Stephanie; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field. PMID:23200243

  7. Adenosine-induced activation of esophageal nociceptors.

    PubMed

    Ru, F; Surdenikova, L; Brozmanova, M; Kollarik, M

    2011-03-01

    Clinical studies implicate adenosine acting on esophageal nociceptive pathways in the pathogenesis of noncardiac chest pain originating from the esophagus. However, the effect of adenosine on esophageal afferent nerve subtypes is incompletely understood. We addressed the hypothesis that adenosine selectively activates esophageal nociceptors. Whole cell perforated patch-clamp recordings and single-cell RT-PCR analysis were performed on the primary afferent neurons retrogradely labeled from the esophagus in the guinea pig. Extracellular recordings were made from the isolated innervated esophagus. In patch-clamp studies, adenosine evoked activation (inward current) in a majority of putative nociceptive (capsaicin-sensitive) vagal nodose, vagal jugular, and spinal dorsal root ganglia (DRG) neurons innervating the esophagus. Single-cell RT-PCR analysis indicated that the majority of the putative nociceptive (transient receptor potential V1-positive) neurons innervating the esophagus express the adenosine receptors. The neural crest-derived (spinal DRG and vagal jugular) esophageal nociceptors expressed predominantly the adenosine A(1) receptor while the placodes-derived vagal nodose nociceptors expressed the adenosine A(1) and/or A(2A) receptors. Consistent with the studies in the cell bodies, adenosine evoked activation (overt action potential discharge) in esophageal nociceptive nerve terminals. Furthermore, the neural crest-derived jugular nociceptors were activated by the selective A(1) receptor agonist CCPA, and the placodes-derived nodose nociceptors were activated by CCPA and/or the selective adenosine A(2A) receptor CGS-21680. In contrast to esophageal nociceptors, adenosine failed to stimulate the vagal esophageal low-threshold (tension) mechanosensors. We conclude that adenosine selectively activates esophageal nociceptors. Our data indicate that the esophageal neural crest-derived nociceptors can be activated via the adenosine A(1) receptor while the placodes

  8. 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

  9. The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei

    PubMed Central

    Haines, M. E.; Johnston, I. R.; Mathias, A. P.; Ridge, D.

    1969-01-01

    1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed. PMID:4311824

  10. Adenosine diphosphate-ribosylation of G-actin by botulinum C2 toxin increases endothelial permeability in vitro.

    PubMed Central

    Suttorp, N; Polley, M; Seybold, J; Schnittler, H; Seeger, W; Grimminger, F; Aktories, K

    1991-01-01

    The endothelial cytoskeleton is believed to play an important role in the regulation of endothelial permeability. We used botulinum C2 toxin to perturb cellular actin and determined its effect on the permeability of endothelial cell monolayers derived from porcine pulmonary arteries. The substrate for botulinum C2 toxin is nonmuscle monomeric actin which becomes ADP-ribosylated. This modified actin cannot participate in actin polymerization and, in addition, acts as a capping protein. Exposure of endothelial cell monolayers to botulinum C2 toxin resulted in a dose- (3-100 ng/ml) and time-dependent (30-120 min) increase in the hydraulic conductivity and decrease in the selectivity of the cell monolayers. The effects of C2 toxin were accompanied by a time- and dose-dependent increase in ADP-ribosylatin of G-actin. G-Actin content increased and F-actin content decreased time- and dose-dependently in C2 toxin-treated endothelial cells. Phalloidin which stabilizes filamentous actin prevented the effects of botulinum C2 toxin on endothelial permeability. Botulinum C2 toxin induced interendothelial gaps. The effects occurred in the absence of overt cell damage and were not reversible within 2 h. The data suggest that the endothelial microfilament system is important for the regulation of endothelial permeability. Images PMID:2022729

  11. Pyrimidine starvation induced by adenosine in fibroblasts and lymphoid cells: role of adenosine deaminase.

    PubMed

    Green, H; Chan, T

    1973-11-23

    In the presence of 10(-4) to 10(-5) molar adenosine, established cell lines of fibroblastic or lymphoid origin die of pyrimidine starvation. Less than lethal concentrations inhibit cell growth. Over a broad concentration range, the effects of adenosine are prevented by providing a suitable pyrimidine source. We suggest that the recently described immune deficiency disease associated with absence of adenosine deaminase may be the result of pyrimidine starvation induced by adenosine nucleotides in cells of the lymphoid system. PMID:4795749

  12. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  13. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  14. Silver indium diphosphate, AgInP(2)O(7).

    PubMed

    Zouihri, Hafid; Saadi, Mohamed; Jaber, Boujemaa; El Ammari, Lehcen

    2010-01-01

    Polycrystalline material of the title compound, AgInP(2)O(7), was synthesized by traditional high-temperature solid-state methods and single crystals were grown from the melt of a mixture of AgInP(2)O(7) and B(2)O(3) as flux in a platinium crucible. The structure consists of InO(6) octa-hedra, which are corner-shared to PO(4) tetra-hedra into a three-dimensional network with hexa-gonal channels running parallel to the c axis. The silver cation, located in the channel, is bonded to seven O atoms of the [InP(2)O(7)] framework with Ag-O distances ranging from 2.370 (2) to 3.015 (2) Å. The P(2)O(7) diphosphate anion is characterized by a P-O-P angle of 137.27 (9) and a nearly eclipsed conformation. AgInP(2)O(7) is isotypic with the M(I)FeP(2)O(7) (M(I) = Na, K, Rb, Cs and Ag) diphosphate family. PMID:21522510

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

    PubMed Central

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

    1997-01-01

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

  16. Structure and Function of a "Head-to-Middle" Prenyltransferase: Lavandulyl Diphosphate Synthase.

    PubMed

    Liu, Meixia; Chen, Chun-Chi; Chen, Lu; Xiao, Xiansha; Zheng, Yingying; Huang, Jian-Wen; Liu, Weidong; Ko, Tzu-Ping; Cheng, Ya-Shan; Feng, Xinxin; Oldfield, Eric; Guo, Rey-Ting; Ma, Yanhe

    2016-04-01

    We report the first X-ray structure of the unique "head-to-middle" monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase. PMID:26922900

  17. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    PubMed Central

    Korhonen, Heidi J; Bolt, Hannah L

    2015-01-01

    Summary Tris[bis(triphenylphosphoranylidene)ammonium] pyrophosphate (PPN pyrophosphate) was used in the SN2 displacements of the tosylate ion from 5’-tosylnucleosides to afford nucleoside-5’-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5’-diphosphates from crude reaction mixtures. PMID:25977720

  18. Characterization of three novel isoprenyl diphosphate synthases from the terpenoid rich mango fruit.

    PubMed

    Kulkarni, Ram; Pandit, Sagar; Chidley, Hemangi; Nagel, Raimund; Schmidt, Axel; Gershenzon, Jonathan; Pujari, Keshav; Giri, Ashok; Gupta, Vidya

    2013-10-01

    Mango (cv. Alphonso) is popular due to its highly attractive, terpenoid-rich flavor. Although Alphonso is clonally propagated, its fruit-flavor composition varies when plants are grown in different geo-climatic zones. Isoprenyl diphosphate synthases catalyze important branch-point reactions in terpenoid biosynthesis, providing precursors for common terpenoids such as volatile terpenes, sterols and carotenoids. Two geranyl diphosphate synthases and a farnesyl diphosphate synthase were isolated from Alphonso fruits, cloned for recombinant expression and found to produce the respective products. Although, one of the geranyl diphosphate synthases showed high sequence similarity to the geranylgeranyl diphosphate synthases, it did not exhibit geranylgeranyl diphosphate synthesizing activity. When modeled, this geranyl diphosphate synthase and farnesyl diphosphate synthase structures were found to be homologous with the reference structures, having all the catalytic side chains appropriately oriented. The optimum temperature for both the geranyl diphosphate synthases was 40 °C and that for farnesyl diphosphate synthase was 25 °C. This finding correlated well with the dominance of monoterpenes in comparison to sesquiterpenes in the fruits of Alphonso mango in which the mesocarp temperature is higher during ripening than development. The absence of activity of these enzymes with the divalent metal ion other than Mg(2+) indicated their adaptation to the Mg(2+) rich mesocarp. The typical expression pattern of these genes through the ripening stages of fruits from different cultivation localities depicting the highest transcript levels of these genes in the stage preceding the maximum terpene accumulation indicated the involvement of these genes in the biosynthesis of volatile terpenes. PMID:23911730

  19. Effects of adenosine on intrarenal oxygenation.

    PubMed

    Dinour, D; Brezis, M

    1991-11-01

    Although generally a vasodilator, adenosine vasoconstricts cortical vessels in the kidney, reduces glomerular filtration rate (GFR), and increases medullary blood flow, effects likely to improve the medullary O2 deficiency characteristic of mammalian kidneys. To evaluate a possible role of adenosine in medullary O2 balance, we investigated the effect of adenosine upon cortical and medullary tissue PO2. Adenosine was infused into renal interstitium through chronically implanted capsules. Cortical and medullary PO2 were measured using sensitive Clark-type O2 microelectrodes inserted into kidneys of anesthetized rats at the respective depths of 1.8 and 3.7 mm. Infusion of adenosine (0.1-0.5 mumol/min) increased medullary PO2 from 17 +/- 3 (SE) to 40 +/- 5 mmHG (P less than 0.001) and decreased cortical PO2 from 64 +/- 4 to 47 +/- 3 mmHg (P less than 0.001). After the infusion was stopped, PO2 returned to baseline at both sites. Coadministration of adenosine receptor antagonist 8-phenyltheophylline (0.01 mumol/min) prevented both cortical and medullary effects of adenosine. We concluded that adenosine could play an important protective and regulatory role in renal medullary O2 balance. PMID:1951710

  20. Adenosine Neuromodulation and Traumatic Brain Injury

    PubMed Central

    Lusardi, T.A

    2009-01-01

    Adenosine is a ubiquitous signaling molecule, with widespread activity across all organ systems. There is evidence that adenosine regulation is a significant factor in traumatic brain injury (TBI) onset, recovery, and outcome, and a growing body of experimental work examining the therapeutic potential of adenosine neuromodulation in the treatment of TBI. In the central nervous system (CNS), adenosine (dys)regulation has been demonstrated following TBI, and correlated to several TBI pathologies, including impaired cerebral hemodynamics, anaerobic metabolism, and inflammation. In addition to acute pathologies, adenosine function has been implicated in TBI comorbidities, such as cognitive deficits, psychiatric function, and post-traumatic epilepsy. This review presents studies in TBI as well as adenosine-related mechanisms in co-morbidities of and unfavorable outcomes resulting from TBI. While the exact role of the adenosine system following TBI remains unclear, there is increasing evidence that a thorough understanding of adenosine signaling will be critical to the development of diagnostic and therapeutic tools for the treatment of TBI. PMID:20190964

  1. Halobacterial adenosine triphosphatases and the adenosine triphosphatase from Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Kristjansson, Hordur; Sadler, Martha H.; Hochstein, Lawrence I.

    1986-01-01

    Membranes prepared from various members of the genus Halobacterium contained a Triton X-l00 activated adenosine triphosphatase. The enzyme from Halobacterium saccharovorum was unstable in solutions of low ionic strength and maximally active in the presence of 3.5 M NaCl. A variety of nucleotide triphosphates was hydrolyzed. MgADP, the product of ATP hydrolysis, was not hydrolyzed and was a competitive inhibitor with respect to MgATP. The enzyme from H. saccharovorum was composed of at least 2 and possibly 4 subunits. The 83-kDa and 60-kDa subunits represented about 90 percent of total protein. The 60-kDa subunit reacted with dicyclohexyl-carbodiimide when inhibition was carried out in an acidic medium. The enzyme from H. saccharovorum, possesses properties of an F(1)F(0) as well as an E(1)E(2) ATPase.

  2. Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.

    PubMed

    Cervoni, L; Lascu, I; Xu, Y; Gonin, P; Morr, M; Merouani, M; Janin, J; Giartosio, A

    2001-04-17

    The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications. PMID:11294625

  3. Specific Activation of A3, A2A and A1 Adenosine Receptors in CD73-Knockout Mice Affects B16F10 Melanoma Growth, Neovascularization, Angiogenesis and Macrophage Infiltration

    PubMed Central

    Koszałka, Patrycja; Gołuńska, Monika; Urban, Aleksandra; Stasiłojć, Grzegorz; Stanisławowski, Marcin; Majewski, Marceli; Składanowski, Andrzej C.; Bigda, Jacek

    2016-01-01

    CD73 (ecto-5'-nucleotidase), a cell surface enzyme hydrolyzing AMP to adenosine, was lately demonstrated to play a direct role in tumor progression including regulation of tumor vascularization. It was also shown to stimulate tumor macrophage infiltration. Interstitial adenosine, accumulating in solid tumors due to CD73 enzymatic activity, is recognized as a main mediator regulating the production of pro- and anti-angiogenic factors, but the engagement of specific adenosine receptors in tumor progression in vivo is still poorly researched. We have analyzed the role of high affinity adenosine receptors A1, A2A, and A3 in B16F10 melanoma progression using specific agonists (CCPA, CGS-21680 and IB-MECA, respectively). We limited endogenous extracellular adenosine background using CD73 knockout mice treated with CD73 chemical inhibitor, AOPCP (adenosine α,β-methylene 5’-diphosphate). Activation of any adenosine receptor significantly inhibited B16F10 melanoma growth but only at its early stage. At 14th day of growth, the decrease in tumor neovascularization and MAPK pathway activation induced by CD73 depletion was reversed by all agonists. Activation of A1AR primarily increased angiogenic activation measured by expression of VEGF-R2 on tumor blood vessels. However, mainly A3AR activation increased both the microvessel density and expression of pro-angiogenic factors. All agonists induced significant increase in macrophage tumor infiltration, with IB-MECA being most effective. This effect was accompanied by substantial changes in cytokines regulating macrophage polarization between pro-inflammatory and pro-angiogenic phenotype. Our results demonstrate an evidence that each of the analyzed receptors has a specific role in the stimulation of tumor angiogenesis and confirm significantly more multifaceted role of adenosine in its regulation than was already observed. They also reveal previously unexplored consequences to extracellular adenosine signaling depletion in

  4. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    SciTech Connect

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

  5. Synthesis and Evaluation of Chlorinated Substrate Analogues for Farnesyl Diphosphate Synthase

    PubMed Central

    Heaps, Nicole A.; Poulter, C. Dale

    2011-01-01

    Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a co-substrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of KM3-ClIPP (with DMAPP) and KM3-ClDMAPP (with IPP) were similar to those for IPP and DMAPP, however values of kcat for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate. PMID:21344952

  6. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    PubMed

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273565

  7. 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

  8. 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

  9. Adenosine Kinase: Exploitation for Therapeutic Gain

    PubMed Central

    2013-01-01

    Adenosine kinase (ADK; EC 2.7.1.20) is an evolutionarily conserved phosphotransferase that converts the purine ribonucleoside adenosine into 5′-adenosine-monophosphate. This enzymatic reaction plays a fundamental role in determining the tone of adenosine, which fulfills essential functions as a homeostatic and metabolic regulator in all living systems. Adenosine not only activates specific signaling pathways by activation of four types of adenosine receptors but it is also a primordial metabolite and regulator of biochemical enzyme reactions that couple to bioenergetic and epigenetic functions. By regulating adenosine, ADK can thus be identified as an upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. Consequently, ADK emerges as a rational therapeutic target, and adenosine-regulating drugs have been tested extensively. In recent attempts to improve specificity of treatment, localized therapies have been developed to augment adenosine signaling at sites of injury or pathology; those approaches include transplantation of stem cells with deletions of ADK or the use of gene therapy vectors to downregulate ADK expression. More recently, the first human mutations in ADK have been described, and novel findings suggest an unexpected role of ADK in a wider range of pathologies. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in a multitude of conditions. This review will provide a comprehensive overview of the genetics, biochemistry, and pharmacology of ADK and will then focus on pathologies and therapeutic interventions. Challenges to translate ADK-based therapies into clinical use will be discussed critically. PMID:23592612

  10. Gas-phase protonation thermochemistry of adenosine.

    PubMed

    Touboul, David; Bouchoux, Guy; Zenobi, Renato

    2008-09-18

    The goal of this work was to obtain a detailed insight on the gas-phase protonation energetic of adenosine using both mass spectrometric experiments and quantum chemical calculations. The experimental approach used the extended kinetic method with nanoelectrospray ionization and collision-induced dissociation tandem mass spectrometry. This method provides experimental values for proton affinity, PA(adenosine) = 979 +/- 1 kJ.mol (-1), and for the "protonation entropy", Delta p S degrees (adenosine) = S degrees (adenosineH +) - S degrees (adenosine) = -5 +/- 5 J.mol (-1).K (-1). The corresponding gas-phase basicity is consequently equal to: GB(adenosine) = 945 +/- 2 kJ.mol (-1) at 298K. Theoretical calculations conducted at the B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d,p) level, including 298 K enthalpy correction, predict a proton affinity value of 974 kJ.mol (-1) after consideration of isodesmic proton transfer reactions with pyridine as the reference base. Moreover, computations clearly showed that N3 is the most favorable protonation site for adenosine, due to a strong internal hydrogen bond involving the hydroxyl group at the 2' position of the ribose sugar moiety, unlike observations for adenine and 2'-deoxyadenosine, where protonation occurs on N1. The existence of negligible protonation entropy is confirmed by calculations (theoretical Delta p S degrees (adenosine) approximately -2/-3 J.mol (-1).K (-1)) including conformational analysis and entropy of hindered rotations. Thus, the calculated protonation thermochemical properties are in good agreement with our experimental measurements. It may be noted that the new PA value is approximately 10 kJ.mol (-1) lower than the one reported in the National Institute of Standards and Technology (NIST) database, thus pointing to a correction of the tabulated protonation thermochemistry of adenosine. PMID:18720985

  11. Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase.

    PubMed

    Kojima, N; Sitthithaworn, W; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Sankaw, U

    2000-07-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs. PMID:10923851

  12. Adenosine triphosphate inhibition of yeast trehalase.

    PubMed

    Panek, A D

    1969-09-01

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

  13. Activation and Inhibition of Ribulose 1,5-Diphosphate Carboxylase by 6-Phosphogluconate 1

    PubMed Central

    Chu, Douglas K.; Bassham, J. A.

    1973-01-01

    Ribulose 1,5-diphosphate carboxylase, when activated by preincubation with 1 mm bicarbonate and 10 mm MgCl2 in the absence of ribulose 1,5-diphosphate, remains activated for 20 minutes or longer after reaction is initiated by addition of ribulose diphosphate. If as little as 50 μm 6-phosphogluconate is added during this preincubation period, 5 minutes before the start of the reaction, a further 188% activation is observed. However, addition of 6-phosphogluconate at the same time or later than addition of ribulose diphosphate, or at any time with 50 mm bicarbonate, gives inhibition of the enzyme activity. Possible relevance of these effects in vivo regulatory effects is discussed. PMID:16658565

  14. Substrate specificity of undecaprenyl diphosphate synthase from the hyperthermophilic archaeon Aeropyrum pernix.

    PubMed

    Mori, Takeshi; Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2013-06-28

    Cis-prenyltransferase from a hyperthermophilic archaeon Aeropyrum pernix was expressed in Escherichia coli and purified for characterization. Properties such as substrate specificity, product chain-length, thermal stability and cofactor requirement were investigated using the recombinant enzyme. In particular, the substrate specificity of the enzyme attracts interest because only dimethylallyl diphosphate and geranylfarnesyl diphosphate, both of which are unusual substrates for known cis-prenyltransferases, are likely available as an allylic primer substrate in A. pernix. From the enzymatic study, the archaeal enzyme was shown to be undecaprenyl diphosphate synthase that has anomalous substrate specificity, which results in a preference for geranylfarnesyl diphosphate. This means that the product of the enzyme, which is probably used as the precursor of the glycosyl carrier lipid, would have an undiscovered structure. PMID:23726912

  15. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

    Holtzman, S.G.; Mante, S.; Minneman, K.P. )

    1991-01-01

    Caffeine is a competitive antagonist at adenosine receptors. Receptor up-regulation during chronic drug treatment has been proposed to be the mechanism of tolerance to the behavioral stimulant effects of caffeine. This study reassessed the role of adenosine receptors in caffeine tolerance. Separate groups of rats were given scheduled access to drinking bottles containing plain tap water or a 0.1% solution of caffeine. Daily drug intake averaged 60-75 mg/kg and resulted in complete tolerance to caffeine-induced stimulation of locomotor activity, which could not be surmounted by increasing the dose of caffeine. 5'-N-ethylcarboxamidoadenosine (0.001-1.0 mg/kg) dose dependently decreased the locomotor activity of caffeine-tolerant rats and their water-treated controls but was 8-fold more potent in the latter group. Caffeine (1.0-10 mg/kg) injected concurrently with 5-N-ethylcarboxamidoadenosine antagonized the decreases in locomotor activity comparably in both groups. Apparent pA2 values for tolerant and control rats also were comparable: 5.05 and 5.11. Thus, the adenosine-antagonist activity of caffeine was undiminished in tolerant rats. The effects of chronic caffeine administration on parameters of adenosine receptor binding and function were measured in cerebral cortex. There were no differences between brain tissue from control and caffeine-treated rats in number and affinity of adenosine binding sites or in receptor-mediated increases (A2 adenosine receptor) and decreases (A1 adenosine receptor) in cAMP accumulation. These results are consistent with theoretical arguments that changes in receptor density should not affect the potency of a competitive antagonist. Experimental evidence and theoretical considerations indicate that up-regulation of adenosine receptors is not the mechanism of tolerance to caffeine-induced stimulation of locomotor activity.

  16. Design and synthesis of phosphonoacetic acid (PPA) ester and amide bioisosters of ribofuranosylnucleoside diphosphates as potential ribonucleotide reductase inhibitors and evaluation of their enzyme inhibitory, cytostatic and antiviral activity.

    PubMed

    Manfredini, Stefano; Solaroli, Nicola; Angusti, Angela; Nalin, Federico; Durini, Elisa; Vertuani, Silvia; Pricl, Sabrina; Ferrone, Marco; Spadari, Silvio; Focher, Federico; Verri, Annalisa; De Clercq, Erik; Balzarini, Jan

    2003-07-01

    Continuing our investigations on inhibitors of ribonucleotide reductase (RNR), the crucial enzyme that catalyses the reduction of ribonucleotides to deoxyribonucleotides, we have now prepared and evaluated 5'-phosphonoacetic acid, amide and ester analogues of adenosine, uridine and cytidine with the aim to verify both substrate specificity and contribution to biological activity of diphosphate mimic moieties. A molecular modelling study has been conducted on the RNR R1 subunit, in order to verify the possible interaction of the proposed bioisosteric moieties. The study compounds were finally tested on the recombinant murine RNR showing a degree of inhibition that ranged from 350 microM for the UDP analogue 5'-deoxy-5'-N-(phosphon-acetyl)uridine sodium salt (amide) to 600 microM for the CDP analogue 5'-O-[(diethyl-phosphon)acetyl]cytidine (ester). None of the tested compounds displayed noteworthy cytostatic activity at 100-500 microM concentrations, whereas ADP analogue 5'-N-[(diethyl-phosphon) acetyl]adenosine (amide) and 5'-deoxy-5'-N-(phosphon-acetyl)adenosine sodium salt (amide) showed a moderate inhibitory activity (EC50: 48 microM) against HSV-2 and a modest inhibitory activity (EC50: 110 microM) against HIV-1, respectively. PMID:14582847

  17. Regulation of Lymphocyte Function by Adenosine

    PubMed Central

    Linden, Joel; Cekic, Caglar

    2014-01-01

    Adenosine regulates the interaction between lymphocytes and the vasculature and is important for controlling lymphocyte trafficking in response to tissue injury or infection. Adenosine can blunt the effects of T cell receptor (TCR) activation primarily by activating adenosine A2A receptors (A2AR) and signaling via cyclic AMP and protein kinase A (PKA). PKA reduces proximal TCR signaling by phosphorylation of C-terminal Src kinase (Csk), nuclear factor of activated T cells (NF-AT) and cyclic AMP response element binding protein (CREB). PKA activation can either enhance or inhibit the survival of T cells depending on the strength and duration of signaling. Inducible enzymes such as CD73 and CD39 regulate adenosine formation and degradation in vivo. The extravasation of lymphocytes through blood vessels is influenced by A2AR-mediated suppression of Intercellular Adhesion Molecule 1 (ICAM) expression on lymphocytes and diminished production of IFNγ and IFNγ-inducible chemokines that are chemotactic to activated lymphocytes. Adenosine also decreases the barrier function of vascular endothelium by activating A2BRs. In sum, adenosine signaling is influenced by tissue inflammation and injury through induction of receptors and enzymes and has generally inhibitory effects on lymphocyte migration into inflamed tissues due to PKA-mediated effects on adhesion molecules, IFNγ production and endothelial barrier function. PMID:22772752

  18. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    SciTech Connect

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  19. Product Rearrangement from Altering a Single Residue in the Rice syn-Copalyl Diphosphate Synthase.

    PubMed

    Potter, Kevin C; Jia, Meirong; Hong, Young J; Tantillo, Dean; Peters, Reuben J

    2016-03-01

    Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism. PMID:26878189

  20. Adenosine receptor agonists attenuate and adenosine receptor antagonists exacerbate opiate withdrawal signs.

    PubMed

    Kaplan, G B; Sears, M T

    1996-01-01

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. Adenosine receptors and their functions have been shown to be regulated by chronic opiate treatment. This study examines the role of adenosine receptors in the expression of opiate withdrawal behaviors. The effects of single doses of parenterally administered adenosine receptor subtype-selective agonists and antagonists on opiate withdrawal signs in morphine-dependent mice were measured. Mice received subcutaneous morphine pellet treatment for 72 h and then underwent naloxone-precipitated withdrawal after pretreatment with adenosinergic agents. Adenosine agonists attenuated different opiate withdrawal signs. The A1 agonist R-N6(phenylisopropyl)adenosine (0, 0.01, 0.02 mg/kg, IP) significantly reduced wet dog shakes and withdrawal diarrhea, while the A2a-selective agonist 2-p-(2-carboxethyl)phenylethylamino-5'-N-ethylcarboxamido adenosine or CGS 21680 (0, 0.01, 0.05 mg/kg, IP) significantly inhibited teeth chattering and forepaw treads. Adenosine receptor antagonists enhanced different opiate withdrawal signs. The adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (0, 1, 10 mg/kg, IP) significantly increased weight loss and the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (0, 1 and 10 mg/kg, IP) enhanced wet dog shakes and withdrawal diarrhea. Treatment effects of adenosinergic agents were not due to nonspecific motor effects, as demonstrated by activity monitoring studies. These results support a role for adenosine receptors in the expression of opiate withdrawal and suggest the potential utility of adenosine agonists in its treatment. PMID:8741956

  1. Potent Radical-Scavenging Activities of Thiamin and Thiamin Diphosphate

    PubMed Central

    Okai, Yasuji; Higashi-Okai, Kiyoka; F. Sato, Eisuke; Konaka, Ryusei; Inoue, Masayasu

    2007-01-01

    Various radical-scavenging activities of thiamin and thiamin diphosphate (TDP) were found in some in vitro experiments. Thiamin and TDP caused considerable suppressive effects on superoxide generation in hypoxanthine and xanthine oxidase system which was measured by a sensitive chemiluminescence method using 2-methyl-6-[p-methylphenyl]-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA), and their 50% inhibition (IC50) values were estimated to be 158 and 56 µM, respectively. They also showed the significant suppression against hydroperoxide generation derived from oxidized linoleic acid which was estimated by aluminum chloride method and their IC50 values were calculated to be 260 and 46 µM. They further prevented the oxygen radical generation in opsonized zymosan-stimulated human blood neutrophils which was shown by chemiluminescence method using luminol, and their IC50 values were calculated to be 169 and 38 µM. In contrast, they caused weak but significantly suppressive effects on the hydroxyl radical generation by Fenton reaction which was measured by electric spin resonance (ESR) method, their IC50 values were calculated to be 8.45 and 1.46 mM respectively. These results strongly suggest a possibility that thiamin and TDP play as radical scavengers in cell-free and cellular systems. PMID:18437212

  2. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    SciTech Connect

    Terekhova, I.V.; Chernyad'ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  3. Effects of cytidine diphosphate choline on rats with memory deficits.

    PubMed

    Petkov, V D; Kehayov, R A; Mosharrof, A H; Petkov, V V; Getova, D; Lazarova, M B; Vaglenova, J

    1993-08-01

    The effects of cytidine diphosphate choline (CDP-choline, CAS 987-78-0) on learning and memory in rats with memory deficits were examined using behavioral methods of active avoidance with punishment reinforcement (shuttle-box), passive avoidance with punishment reinforcement (step-through and step-down), and active avoidance with positive (alimentary) reinforcement (staircase-maze). In the majority of experiments CDP-choline was applied orally at doses of 10-50 or 100 mg/kg daily for 7 days before the training session. The experiments were carried out on young-adult (aged 5 months) and old (aged 22 months) rats and on rats with a low capability for retention of learned behavior. Memory deficits were induced by the muscarinic cholinoceptor antagonist scopolamine (in young and old rats and mice), by the alpha 2-adrenoceptor agonist clonidine, by electroconvulsive shock, and by hypoxy. Memory deficits were also induced in rats offspring of dams that had been exposed to alcohol during pregnancy and lactation. The results suggest that CDP-choline acts as a memory-enhancing drug and that its effect is particularly pronounced in animals with memory deficits. PMID:8216435

  4. Preliminary study of irradiation effects on thorium phosphate-diphosphate

    NASA Astrophysics Data System (ADS)

    Pichot, E.; Dacheux, N.; Emery, J.; Chaumont, J.; Brandel, V.; Genet, M.

    2001-03-01

    Thorium phosphate-diphosphate (TPD): Th 4(PO 4) 4P 2O 7 is proposed as a host matrix for the long-term storage of high level radioactive wastes. Indeed, γ-rays, α and β particles due to the incorporated actinides or fission products will certainly produce several effects, particularly structural and chemical modifications, in the host material. In order to investigate these effects, powdered samples were irradiated with 1.5 Gy dose of γ-rays. The formation of PO 32- and POO rad free radicals was detected using electron spin resonance (ESR) and thermoluminescence (TL) methods. These free radicals do not modify the macroscopic properties of the TPD and disappear when the sample is heated at 400°C. The implantation of He + ions of 1.6 MeV (fluence: 10 16 particles cm -2) and Au 3+ ions of 5 MeV (fluence 4×10 15 particles cm -2) causes some damages on the surface of sintered samples. Amorphization and chemical decomposition of the matrix were observed for the dose of 10 15 particles cm -2 and higher when irradiated with Pb 2+ (200 keV) and Au 3+ (5 MeV) ion beams. These effects were evidenced by means of X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS).

  5. 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.

  6. Photoaffinity labeling of A1-adenosine receptors

    SciTech Connect

    Klotz, K.N.; Cristalli, G.; Grifantini, M.; Vittori, S.; Lohse, M.J.

    1985-11-25

    The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for (TH)N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of (TH)N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity ( SVI-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for SVI-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that SVI-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.

  7. Application of the functionalized congener approach to dendrimer-based signaling agents acting through A(2A) adenosine receptors.

    PubMed

    Kim, Yoonkyung; Klutz, Athena M; Hechler, Béatrice; Gao, Zhan-Guo; Gachet, Christian; Jacobson, Kenneth A

    2009-03-01

    As a continued effort to develop multivalent ligands to enhance the pharmacological effects of monomeric drugs, DITC-APEC, a chemically reactive nucleoside A(2A) adenosine receptor (AR) agonist, was employed to derivatize the surface of third-generation (G3) polyamidoamine (PAMAM) dendrimers. The resulting conjugates carried multiple copies of the agonist attached through a thiourea linkage and differed in the number of attachments and in the presence of a fluorophore or additional surface modification. Computer modeling studies suggested that these DITC-APEC-loaded dendrimers extended the overall diameter of the previously reported PAMAM-CGS21680 dendrimer derivatives (Kim et al., Bioconjugate Chem 2008 19:406-411) by ca. 20 A, potentially increasing the conformational flexibility of the appended ligands to achieve optimal geometry for efficient binding at A(2A) ARs. Increased affinity and selectivity in binding in comparison to the CGS21680 conjugate were envisioned, due to the presence of an extended linker, i.e., a dithioureylenephenyl functionality. In vitro radioligand competition experiments showed effective binding of these PAMAM-DITC-APEC dendrimer conjugates at the human A(2A) and A(3) ARs with submicromolar K (i) values and selectivity in comparison to the human A(1) AR. Furthermore, these nucleoside-loaded dendrimers exhibited an A(2A) AR-mediated inhibitory effect on ADP-induced aggregation of human platelets. The present study demonstrates the potential of applying the functionalized congener concept to engineer dendrimer-based multivalent ligands for G protein-coupled receptors. PMID:18600474

  8. Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production.

    PubMed

    Kang, Aram; George, Kevin W; Wang, George; Baidoo, Edward; Keasling, Jay D; Lee, Taek Soon

    2016-03-01

    Branched C5 alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C5 alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene. PMID:26708516

  9. Lithium-cation conductivity and crystal structure of lithium diphosphate

    SciTech Connect

    Voronin, V.I.; Sherstobitova, E.A.; Blatov, V.A.; Shekhtman, G.Sh.

    2014-03-15

    The electrical conductivity of lithium diphosphate Li{sub 4}P{sub 2}O{sub 7} has been measured and jump-like increasing of ionic conductivity at 913 K has been found. The crystal structure of Li{sub 4}P{sub 2}O{sub 7} has been refined using high temperature neutron diffraction at 300–1050 K. At 913 K low temperature triclinic form of Li{sub 4}P{sub 2}O{sub 7} transforms into high temperature monoclinic one, space group P2{sub 1}/n, a=8.8261(4) Å, b=5.2028(4) Å, c=13.3119(2) Å, β=104.372(6)°. The migration maps of Li{sup +} cations based on experimental data implemented into program package TOPOS have been explored. It was found that lithium cations in both low- and high temperature forms of Li{sub 4}P{sub 2}O{sub 7} migrate in three dimensions. Cross sections of the migrations channels extend as the temperature rises, but at the phase transition point have a sharp growth showing a strong “crystal structure – ion conductivity” correlation. -- Graphical abstract: Crystal structure of Li{sub 4}P{sub 2}O{sub 7} at 950 K. Red balls represent oxygen atoms; black lines show Li{sup +} ion migration channels in the layers perpendicular to [001] direction. Highlights: • Structure of Li{sub 4}P{sub 2}O{sub 7} has been refined using high temperature neutron diffraction. • At 913 K triclinic form of Li{sub 4}P{sub 2}O{sub 7} transforms into high temperature monoclinic one. • The migration maps of Li{sup +} implemented into program package TOPOS have been explored. • Cross sections of the migrations channels at the phase transition have a sharp growth.

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

    PubMed Central

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

    1994-01-01

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

  11. Nucleoside transporter expression and adenosine uptake in the rat cochlea.

    PubMed

    Khan, Abdul F; Thorne, Peter R; Muñoz, David J B; Wang, Carol J H; Housley, Gary D; Vlajkovic, Srdjan M

    2007-02-12

    Even though extracellular adenosine plays multiple roles in the cochlea, the mechanisms that control extracellular adenosine concentrations in this organ are unclear. This study investigated the expression of nucleoside transporters and adenosine uptake in the rat cochlea. Reverse transcription-polymerase chain reaction revealed the expression of mRNA transcripts for two equilibrative (ENT1 and ENT2) and two concentrative (CNT1 and CNT2) nucleoside transporters. Exogenous adenosine perfused through the cochlear perilymphatic compartment was taken up by cells lining the compartment. Adenosine uptake was sensitive to changes in extracellular Na concentrations and inhibited by nitrobenzylthioinosine (an adenosine uptake blocker). The study suggests that the bi-directional nucleoside transport supports the uptake and recycling of purines and regulates the activation of adenosine receptors by altering adenosine concentrations in cochlear fluid spaces. PMID:17314663

  12. Novel adenosine receptors in rat hippocampus identification and characterization

    SciTech Connect

    Chin, J.H.; Mashman, W.E.; DeLorenzo, R.J.

    1985-05-06

    2-chloro(/sup 3/H)adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a K/sub D/ of 4.7 ..mu..M and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability. 50 references, 2 figures, 1 table.

  13. A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity.

    PubMed

    Rivera-Perez, Crisalejandra; Nyati, Pratik; Noriega, Fernando G

    2015-09-01

    Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg(2+) ions lead to the production of FPP, while the presence of Co(2+) ions lead to geranyl diphosphate (GPP) production. In the presence of Mg(2+) the AaFPPS affinity for allylic substrates is GPP > DMAPP > IPP. These results suggest that AaFPPS displays "catalytic promiscuity", changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways. PMID:26188328

  14. Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

    PubMed Central

    Snyder, F F; Mendelsohn, J; Seegmiller, J E

    1976-01-01

    The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 muM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes. Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemagglutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 muM adenosine, and deamination at concentrations greater than 5 muM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine

  15. Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus

    PubMed Central

    Duong-ly, Krisna C.; Schoeffield, Andrew J.; Pizarro-Dupuy, Mario A.; Zarr, Melissa; Pineiro, Silvia A.; Amzel, L. Mario; Gabelli, Sandra B.

    2015-01-01

    Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from Bdellovibrio bacteriovorus–that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases. PMID:26524597

  16. Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters.

    PubMed

    Lynge, J; Juel, C; Hellsten, Y

    2001-12-01

    1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the

  17. Tenofovir diphosphate concentrations and prophylactic effect in a macaque model of rectal simian HIV transmission

    PubMed Central

    Anderson, Peter L.; Glidden, David V.; Bushman, Lane R.; Heneine, Walid; García-Lerma, J. Gerardo

    2014-01-01

    Objectives This study evaluated the relationship between intracellular tenofovir diphosphate concentrations in peripheral blood mononuclear cells and prophylactic efficacy in a macaque model for HIV pre-exposure prophylaxis (PrEP). Methods Macaques were challenged with simian HIV (SHIV) via rectal inoculation once weekly for up to 14 weeks. A control group (n = 34) received no drug, a second group (n = 6) received oral tenofovir disoproxil fumarate/emtricitabine 3 days before each virus challenge and a third group (n = 6) received the same dosing plus another dose 2 h after virus challenge. PBMCs were collected just before each weekly virus challenge. The relationship between tenofovir diphosphate in PBMCs and prophylactic efficacy was assessed with a Cox proportional hazards model. Results The percentages of animals infected in the control, one-dose and two-dose groups were 97, 83 and 17, respectively. The mean (SD) steady-state tenofovir diphosphate concentration (fmol/106 cells) was 15.8 (7.6) in the one-dose group and 30.7 (10.1) in the two-dose group. Each 5 fmol tenofovir diphosphate/106 cells was associated with a 40% (95% CI 17%–56%) reduction in risk of SHIV acquisition, P = 0.002. The tenofovir diphosphate concentration associated with a 90% reduction in risk (EC90) was 22.6 fmol/106 cells (95% CI 13.8–60.8). Conclusions The prophylactic EC90 for tenofovir diphosphate identified in macaques exposed rectally compares well with the EC90 previously identified in men who have sex with men (MSM; 16 fmol/106 cells, 95% CI 3–28). These results highlight the relevance of this model to inform human PrEP studies of oral tenofovir disoproxil fumarate/emtricitabine for MSM. PMID:24862094

  18. Adenosine reagent-free detection by co-immobilization of adenosine deaminase and phenol red on an optical biostrip.

    PubMed

    Bartzoka, Foteini; Venetsanou, Katerina; Clonis, Yannis

    2015-01-01

    Adenosine detection in human serum is important because this ribonucleoside has established clinical applications, modulating many physiological processes. Furthermore, a simple and cheap detection method is useful in adenosine production processes. Adenosine can be determined enzymatically using either S-adenosyl-homocysteine hydrolase and (3) [H]-adenosine, or adenosine kinase combined with GTP and luciferase, or an amperometric biosensor carrying adenosine deaminase (ADA), purine nucleoside phosphorylase, and xanthine oxidase. We developed a simple and cheap method relying on a transparent biostrip bearing ADA and the indicator phenol red (PR), co-immobilized to polyacrylamide, itself chemically adhered to a derivatized glass strip. The ADA-catalyzed conversion of adenosine to inosine and ammonia leads to a local pH alteration, changing the absorbance maximum of PR (from 425 to 567 nm), which is measured optically. The biostrip shows an analytical range 0.05-1.5 mM adenosine and is reusable when stored at 4 °C. When the biostrip was tested with serum, spiked with adenosine (70 and 100 μM), and filtered for protein and adenosine phosphates depletion, it showed good adenosine recovery. In summary, we show the proof-of-concept that adenosine can be determined reagent-free, at moderate sensitivity on an easy to construct, cheap, and reusable biostrip, based on commercially available molecular entities. PMID:25293641

  19. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    SciTech Connect

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. )

    1993-07-01

    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  20. Internalization and desensitization of adenosine receptors

    PubMed Central

    Klaasse, Elisabeth C.; de Grip, Willem J.; Beukers, Margot W.

    2007-01-01

    Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A1, A2A, A2B and A3 receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2AR and A2BR undergo much faster downregulation, usually shorter than 1 h. The A3R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed. PMID:18368531

  1. Neuroprotective effects of adenosine deaminase in the striatum.

    PubMed

    Tamura, Risa; Ohta, Hiroyuki; Satoh, Yasushi; Nonoyama, Shigeaki; Nishida, Yasuhiro; Nibuya, Masashi

    2016-04-01

    Adenosine deaminase (ADA) is a ubiquitous enzyme that catabolizes adenosine and deoxyadenosine. During cerebral ischemia, extracellular adenosine levels increase acutely and adenosine deaminase catabolizes the increased levels of adenosine. Since adenosine is a known neuroprotective agent, adenosine deaminase was thought to have a negative effect during ischemia. In this study, however, we demonstrate that adenosine deaminase has substantial neuroprotective effects in the striatum, which is especially vulnerable during cerebral ischemia. We used temporary oxygen/glucose deprivation (OGD) to simulate ischemia in rat corticostriatal brain slices. We used field potentials as the primary measure of neuronal damage. For stable and efficient electrophysiological assessment, we used transgenic rats expressing channelrhodopsin-2, which depolarizes neurons in response to blue light. Time courses of electrically evoked striatal field potential (eFP) and optogenetically evoked striatal field potential (optFP) were recorded during and after oxygen/glucose deprivation. The levels of both eFP and optFP decreased after 10 min of oxygen/glucose deprivation. Bath-application of 10 µg/ml adenosine deaminase during oxygen/glucose deprivation significantly attenuated the oxygen/glucose deprivation-induced reduction in levels of eFP and optFP. The number of injured cells decreased significantly, and western blot analysis indicated a significant decrease of autophagic signaling in the adenosine deaminase-treated oxygen/glucose deprivation slices. These results indicate that adenosine deaminase has protective effects in the striatum. PMID:26746865

  2. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors.

    PubMed

    Gao, Zhan-Guo; Mamedova, Liaman K; Chen, Peiran; Jacobson, Kenneth A

    2004-11-15

    The affinity and efficacy at four subtypes (A(1), A(2A), A(2B) and A(3)) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N(6)-position, several 2-substituents were found to be critical structural determinants for the A(3)AR activation. The following adenosine 2-ethers were moderately potent partial agonists (K(i), nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A(3)AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)adenosine as an A(3)AR antagonist right-shifted the concentration-response curve for the inhibition by NECA of cyclic AMP accumulation with a K(B) value of 212 nM, which is similar to its binding affinity (K(i) = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A(1)AR in comparison to the A(3)AR, but fully efficacious, with binding K(i) values over 100 nM. The 2-phenylethyl moiety resulted in higher A(3)AR affinity (K(i) in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (K(i) = 3.8 nM) was found to be the most potent and selective (>50-fold) A(2A) agonist in this series. Mixed A(2A)/A(3)AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A(2B)AR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC(50) = 1.4 microM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC(50) = 1.8 microM) were found to be relatively potent A(2B) agonists, although less potent than NECA (EC(50) = 140 nM). PMID:15476669

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

    PubMed

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

    2016-03-15

    Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes. PMID:26409025

  4. Chrysanthemyl Diphosphate Synthase Operates in Planta as a Bifunctional Enzyme with Chrysanthemol Synthase Activity*

    PubMed Central

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12–0.16 μg h−1 g−1 fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  5. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    PubMed

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate. PMID:25378387

  6. Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate.

    PubMed

    Tornheim, K; Lowenstein, J M

    1976-12-10

    Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate. PMID:12161

  7. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors

    PubMed Central

    Gao, Zhan-Guo; Mamedova, Liaman K.; Chen, Peiran; Jacobson, Kenneth A.

    2012-01-01

    The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM). PMID:15476669

  8. Silk polymer-based adenosine release: therapeutic potential for epilepsy.

    PubMed

    Wilz, Andrew; Pritchard, Eleanor M; Li, Tianfu; Lan, Jing-Quan; Kaplan, David L; Boison, Detlev

    2008-09-01

    Adenosine augmentation therapies (AAT) make rational use of the brain's own adenosine-based seizure control system and hold promise for the therapy of refractory epilepsy. In an effort to develop an AAT compatible with future clinical application, we developed a novel silk protein-based release system for adenosine. Adenosine releasing brain implants with target release doses of 0, 40, 200, and 1000ng adenosine per day were prepared by embedding adenosine containing microspheres into nanofilm-coated silk fibroin scaffolds. In vitro, the respective polymers released 0, 33.4, 170.5, and 819.0ng adenosine per day over 14 days. The therapeutic potential of the implants was validated in a dose-response study in the rat model of kindling epileptogenesis. Four days prior to the onset of kindling, adenosine releasing polymers were implanted into the infrahippocampal cleft and progressive acquisition of kindled seizures was monitored over a total of 48 stimulations. We document a dose-dependent retardation of seizure acquisition. In recipients of polymers releasing 819ng adenosine per day, kindling epileptogenesis was delayed by one week corresponding to 18 kindling stimulations. Histological analysis of brain samples confirmed the correct location of implants and electrodes. We conclude that silk-based delivery of around 1000ng adenosine per day is a safe and efficient strategy to suppress seizures. PMID:18514814

  9. Adenosine Signaling During Acute and Chronic Disease States

    PubMed Central

    Karmouty-Quintana, Harry; Xia, Yang; Blackburn, Michael R.

    2013-01-01

    Adenosine is a signaling nucleoside that is produced following tissue injury, particularly injury involving ischemia and hypoxia. The production of extracellular adenosine and its subsequent signaling through adenosine receptors plays an important role in orchestrating injury responses in multiple organs. There are four adenosine receptors that are widely distributed on immune, epithelial, endothelial, neuronal and stromal cells throughout the body. Interestingly, these receptors are subject to altered regulation following injury. Studies in mouse models and human cells and tissues have identified that the production of adenosine and its subsequent signaling through its receptors plays largely beneficial roles in acute disease states, with the exception of brain injury. In contrast, if elevated adenosine levels are sustained beyond the acute injury phase, adenosine responses can become detrimental by activating pathways that promote tissue injury and fibrosis. Understanding when during the course of disease adenosine signaling is beneficial as opposed to detrimental and defining the mechanisms involved will be critical for the advancement of adenosine based therapies for acute and chronic diseases. The purpose of this review is to discuss key observations that define the beneficial and detrimental aspects of adenosine signaling during acute and chronic disease states with an emphasis on cellular processes such as inflammatory cell regulation, vascular barrier function and tissue fibrosis. PMID:23340998

  10. Adenosine thallium 201 myocardial perfusion scintigraphy

    SciTech Connect

    Verani, M.S. )

    1991-07-01

    Pharmacologic coronary vasodilation as an adjunct to myocardial perfusion imaging has become increasingly important in the evaluation of patients with coronary artery disease, in view of the large number of patients who cannot perform an adequate exercise test or in whom contraindications render exercise inappropriate. Adenosine is a very potent coronary vasodilator and when combined with thallium 201 scintigraphy produces images of high quality, with the added advantages of a very short half-life (less than 10 seconds) and the ability to adjust the dose during the infusion, which may enhance safety and curtail the duration of side effects. The reported sensitivity and specificity of adenosine thallium 201 scintigraphy for the detection of coronary artery disease are high and at least comparable with imaging after exercise or dipyridamole administration. 23 refs.

  11. Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases

    PubMed Central

    Keeling, Christopher I.; Chiu, Christine C.; Aw, Tidiane; Li, Maria; Henderson, Hannah; Tittiger, Claus; Weng, Hong-Biao; Blomquist, Gary J.; Bohlmann, Joerg

    2013-01-01

    The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon–carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors. PMID:24167290

  12. Prevalence of unidirectional Na+-dependent adenosine transport and altered potential for adenosine generation in diabetic cardiac myocytes.

    PubMed

    Podgorska, M; Kocbuch, K; Grden, M; Szutowicz, A; Pawelczyk, T

    2006-05-01

    Adenosine is an important physiological regulator of the cardiovascular system. The goal of our study was to assess the expression level of nucleoside transporters (NT) in diabetic rat cardiomyocytes and to examine the activities of adenosine metabolizing enzymes. Isolated rat cardiomyocytes displayed the presence of detectable amounts of mRNA for ENT1, ENT2, CNT1, and CNT2. Overall adenosine (10 microM) transport in cardiomyocytes isolated from normal rat was 36 pmol/mg/min. The expression level of equilibrative transporters (ENT1, ENT2) decreased and of concentrative transporters (CNT1, CNT2) increased in myocytes isolated from diabetic rat. Consequently, overall adenosine transport decreased by 30%, whereas Na(+)-dependent adenosine uptake increased 2-fold, and equilibrative transport decreased by 60%. The activity ratio of AMP deaminase/5'-nucleotidase in cytosol of normal cardiomyocytes was 11 and increased to 15 in diabetic cells. The activity of ecto-5'-nucleotidase increased 2-fold in diabetic cells resulting in a rise of the activity ratio of ecto-5'-nucleotidase/adenosine deaminase from 28 to 56.These results indicate that in rat cardiomyocytes diabetes alters activities of adenosine metabolizing enzymes in such a way that conversion of AMP to IMP is favored in the cytosolic compartment, whereas the capability to produce adenosine extracellularly is increased. This is accompanied by an increased unidirectional Na(+)-dependent uptake of adenosine and significantly reduced bidirectional adenosine transport. PMID:16369729

  13. ATP- and adenosine-mediated signaling in the central nervous system: adenosine stimulates glutamate release from astrocytes via A2a adenosine receptors.

    PubMed

    Nishizaki, Tomoyuki

    2004-02-01

    Adenosine enhanced intracellular Ca(2+) concentrations in astrocytes via A(2a) adenosine receptors involving protein kinase A (PKA) activation. The Ca(2+) rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP(3)-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A(2a) adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A(2a) adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca(2+) release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes. PMID:14978344

  14. Use of adenosine echocardiography for diagnosis of coronary artery disease

    SciTech Connect

    Zoghbi, W.A. )

    1991-07-01

    Two-dimensional echocardiography combined with exercise is sensitive and specific in the detection of coronary artery disease (CAD) by demonstrating transient abnormalities in wall motion. Frequently, however, patients cannot achieve maximal exercise because of various factors. Pharmacologic stress testing with intravenous adenosine was evaluated as a means of detecting CAD in a noninvasive manner. Patients with suspected CAD underwent echocardiographic imaging and simultaneous thallium 201 single-photon emission computed tomography during the intravenous administration of 140 micrograms/kg/min of adenosine. An increase in heart rate, decrease in blood pressure, and increase in double product were observed during adenosine administration. Initial observations revealed that wall motion abnormalities were induced by adenosine in areas of perfusion defects. The adenosine infusion was well tolerated, and symptoms disappeared within 1 to 2 minutes after termination of the infusion. Therefore preliminary observations suggest that adenosine echocardiography appears to be useful in the assessment of CAD.

  15. Characterization of adenosine receptors involved in adenosine-induced bronchoconstriction in allergic rabbits.

    PubMed Central

    el-Hashim, A.; D'Agostino, B.; Matera, M. G.; Page, C.

    1996-01-01

    1. Recent work has suggested that adenosine may be involved in asthma via the activation of A1 receptors. However, the role of the recently cloned A3 receptor in airways is largely unknown. In the present study, we have investigated the role of the A3 receptor in adenosine-induced bronchoconstriction in allergic rabbits. 2. Aerosol challenge of antigen (Ag) immunized rabbits with the adenosine precursor, adenosine 5'-monophosphate (AMP), resulted in a dose-dependent fall in dynamic compliance (Cdyn). The maximum fall in Cdyn in these rabbits was significantly greater than that in litter matched, sham immunized animals (P < 0.05). However, there was no significant difference in the maximum increase in airways resistance (Rt) between Ag and sham immunized rabbits (P > 0.05). 3. Aerosol challenge of Ag immunized rabbits with cyclopentyl-adenosine (CPA) (A1-receptor agonist) elicited a dose-dependent fall in Cdyn in Ag immunized rabbits and the maximum fall in Cdyn in these rabbits was significantly greater than that observed in sham immunized rabbits (P < 0.05). Similarly, CPA induced dose-dependent increases in R1 in Ag immunized rabbits whereas sham immunized rabbits failed to respond to CPA within the same dose range. The maximum increase in RL in Ag immunized rabbits was significantly greater than that of sham immunized rabbits (P < 0.05). 4. Aerosol challenge of either Ag or sham immunized rabbits with the A3 agonist aminophenylethyladenosine (APNEA) did not elicit dose-dependent changes in either RL or Cdyn. Moreover, there was no significant difference in the maximum response, measured by either parameter, between the two animal groups (P > 0.05). 5. These data provide further evidence for a role of the A1 receptor in the airways, but do not support a role for the A3 receptor in adenosine-induced bronchoconstriction in the allergic rabbit. PMID:8937732

  16. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment

    PubMed Central

    Ohta, Akio

    2016-01-01

    Within tumors, some areas are less oxygenated than others. Since their home ground is under chronic hypoxia, tumor cells adapt to this condition by activating aerobic glycolysis; however, this hypoxic environment is very harsh for incoming immune cells. Deprivation of oxygen limits availability of energy sources and induces accumulation of extracellular adenosine in tumors. Extracellular adenosine, upon binding with adenosine receptors on the surface of various immune cells, suppresses pro-inflammatory activities. In addition, signaling through adenosine receptors upregulates a number of anti-inflammatory molecules and immunoregulatory cells, leading to the establishment of a long-lasting immunosuppressive environment. Thus, due to hypoxia and adenosine, tumors can discourage antitumor immune responses no matter how the response was induced, whether it was spontaneous or artificially introduced with a therapeutic intention. Preclinical studies have shown the significance of adenosine in tumor survival strategy by demonstrating tumor regression after inactivation of adenosine receptors, inhibition of adenosine-producing enzymes, or reversal of tissue hypoxia. These promising results indicate a potential use of the inhibitors of the hypoxia–adenosine pathway for cancer immunotherapy. PMID:27066002

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

    PubMed Central

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

    1984-01-01

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

  18. The formation of mono-N-acetylhexosamine derivatives of dolichol diphosphate by pig liver microsomal fractions.

    PubMed Central

    Palamarczyk, G; Hemming, F W

    1975-01-01

    Incubation of pig liver microsomal preparations with UDP-N[U-14C]acetylglucosamine yields a 14C-labelled lipid. The requirement for Mn2+, the pH optimum, time-dependence and the reversibility by UMP of the transferase are reported. Evidence is presented in favour of the lipid being a mixture of dolichol diphosphate N-[14C]acetylglucosamine and dolichol diphosphate N-[14C]acetylmannosamine. Available data suggest that the epimerization takes place while the hexosamine is bound in this lipid-soluble form. The N-acetylmannosamine appeared not be be released into the medium. The subfractionation of the microsomal fraction to separate transferase activity from membrane-bound beta-N-acetylglucosaminidase activity is also reported. Images PLATE 1 PMID:239708

  19. [A pharmacological study of the hepatoprotective activity of fructose-1,6-diphosphate].

    PubMed

    Klouchek, E; Markov, M; Popov, A

    1993-01-01

    A fructose-1,6-diphosphate preparation was tested for hepatoprotective activity through biochemical and morphologic studies in experiments on Wistar rats sustaining D-galactosamine- and paracetamole-induced hepatotoxicity. Findings indicated the modeled hepatic lesions to be readily reproducible, to simulate some characteristics of human liver pathology, and to be suitable for testing substances expected to have hepatoprotective action; intraperitoneal administration of fructose-1,6-diphosphate at a dose of 1000 mg/kg body weight proved moderately protective against liver damage by D-galactosamine; the benefit observed concerned mostly dystrophic and inflammatory changes in the liver; in a number of cases, correlation was noted between biochemical serum parameters and pathomorphologic liver alterations. PMID:7805621

  20. Adenosine: Tipping the balance towards hepatic steatosis and fibrosis

    PubMed Central

    Robson, Simon C.; Schuppan, Detlef

    2010-01-01

    Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the histochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver. PMID:20395005

  1. Enantioselective Inhibition of Squalene Synthase by Aziridine Analogues of Presqualene Diphosphate

    PubMed Central

    Koohang, Ali; Bailey, Jessica L.; Erickson, Hans K.; Owen, David; Poulter, C. Dale

    2013-01-01

    Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates (6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation–reduction sequence of (±)-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldi-methylsilyl ethers. SN2 displacements of the corresponding methanesulfonates with pyrophosphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and methanediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(α-methylene)bishomogeranyl substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute stereochemistry to PSPP proved to be the most potent inhibitor (IC50 1.17 ± 0.08 μM in the presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The other aziridine analogues bearing the N-(α-methylene)bishomogeranyl and N-undecyl substituents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene synthase. PMID:20545375

  2. A cesium copper vanadyl-diphosphate: Synthesis, crystal structure and physical properties

    SciTech Connect

    Shvanskaya, Larisa; Yakubovich, Olga; Bychkov, Andrey; Shcherbakov, Vasiliy; Golovanov, Alexey; Zvereva, Elena; Volkova, Olga; Vasiliev, Alexander

    2015-02-15

    A non-centrosymmetric orthorhombic diphosphate, Cs{sub 2}Cu{sub 1+x}(VO){sub 2−x}(P{sub 2}O{sub 7}){sub 2} (x=0.1) with a=13.7364(2) Å, b=9.2666(2) Å, c=11.5678(2) Å, Z=4, has been isolated. Its 3D framework is built from Cu atoms in square pyramidal and square planar coordination, VO{sub 5} tetragonal pyramids and P{sub 2}O{sub 7} diphosphate groups, sharing vertices. Large channels are fulfilled by cesium atoms. The ESR study reveals a similarity in behaviour of two paramagnetic (Cu and V) subsystems. The temperature dependences of the ESR linewidth and static magnetic susceptibility data present evidences for a cluster type magnetic ordering in the title compound at T⁎=22 K. The weakness of the relevant anomalies reflects presumably obvious Cu{sup 2+} ions and (VO){sup 2+} units disorder in the system. It is supposed that the charge and geometry of the framework are controlled by the Cu{sup 2+}/(VO){sup 2+} ratio; its variation may lead to a design of new materials. - Graphical abstract: A microporous 3D anionic framework of the first copper vanadium-diphosphate Cs{sub 2}Cu{sub 1.1}(VO){sub 1.9}(P{sub 2}O{sub 7}){sub 2}. The similarity in behaviour of Cu and V paramagnetic subsystems revealed by ESR study. - Highlights: • The first copper vanadium-diphosphate Cs{sub 2}Cu{sub 1.1}(VO){sub 1.9}(P{sub 2}O{sub 7}){sub 2} is reported. • A 3D anionic framework is characterized by disorder in distribution of Cu and V atoms. • Structural relations with topologically similar compounds are discussed. • The similarity in behaviour of Cu and V paramagnetic subsystems has been revealed.

  3. Farnesyl diphosphate synthase localizes to the cytoplasm of Trypanosoma cruzi and T. brucei.

    PubMed

    Ferella, Marcela; Li, Zhu-Hong; Andersson, Björn; Docampo, Roberto

    2008-06-01

    The farnesyl diphosphate synthase (FPPS) has previously been characterized in trypanosomes as an essential enzyme for their survival and as the target for bisphosphonates, drugs that are effective both in vitro and in vivo against these parasites. Enzymes from the isoprenoid pathway have been assigned to different compartments in eukaryotes, including trypanosomatids. We here report that FPPS localizes to the cytoplasm of both Trypanosoma cruzi and T. brucei, and is not present in other organelles such as the mitochondria and glycosomes. PMID:18406406

  4. Pharmacokinetic study of the structural components of adenosine diphosphate-encapsulated liposomes coated with fibrinogen γ-chain dodecapeptide as a synthetic platelet substitute.

    PubMed

    Taguchi, Kazuaki; Ujihira, Hayato; Ogaki, Shigeru; Watanabe, Hiroshi; Fujiyama, Atsushi; Doi, Mami; Okamura, Yosuke; Takeoka, Shinji; Ikeda, Yasuo; Handa, Makoto; Otagiri, Masaki; Maruyama, Toru

    2013-08-01

    Fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV, H12)-coated, ADP-encapsulated liposomes [H12-(ADP)-liposomes] were developed as a synthetic platelet alternative that specifically accumulates at bleeding sites as the result of interactions with activated platelets via glycoprotein IIb/IIIa and augments platelet aggregation by releasing ADP. The aim of this study is to characterize the pharmacokinetic properties of H12-(ADP)-liposomes and structural components in rats, and to predict the blood retention of H12-(ADP)-liposomes in humans. With use of H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane cholesterol were radiolabeled with (14)C and (3)H, respectively, it was found that the time courses for the plasma concentration curves of (14)C and (3)H radioactivity showed that the H12-(ADP)-liposomes remained intact in the blood circulation for up to 24 hours after injection, and were mainly distributed to the liver and spleen. However, the (14)C and (3)H radioactivity of H12-(ADP)-liposomes disappeared from organs within 7 days after injection. The encapsulated ADP was metabolized to allantoin, which is the final metabolite of ADP in rodents, and was mainly eliminated in the urine, whereas the cholesterol was mainly eliminated in feces. In addition, the half-life of the H12-(ADP)-liposomes in humans was predicted to be approximately 96 hours from pharmacokinetic data obtained for mice, rats, and rabbits using an allometric equation. These results suggest that the H12-(ADP)-liposome has potential with proper pharmacokinetic and acceptable biodegradable properties as a synthetic platelet substitute. PMID:23735758

  5. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

  6. Molecular Cloning and Characterization of a Geranyl Diphosphate-Specific Aromatic Prenyltransferase from Lemon1[W

    PubMed Central

    Munakata, Ryosuke; Inoue, Tsuyoshi; Koeduka, Takao; Karamat, Fazeelat; Olry, Alexandre; Sugiyama, Akifumi; Takanashi, Kojiro; Dugrand, Audray; Froelicher, Yann; Tanaka, Ryo; Uto, Yoshihiro; Hori, Hitoshi; Azuma, Jun-Ichi; Hehn, Alain; Bourgaud, Frédéric; Yazaki, Kazufumi

    2014-01-01

    Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species. PMID:25077796

  7. 6- and 14-Fluoro farnesyl diphosphate: mechanistic probes for the reaction catalysed by aristolochene synthase.

    PubMed

    Miller, David J; Yu, Fanglei; Knight, David W; Allemann, Rudolf K

    2009-03-01

    The catalytic mechanism of the enzyme aristolochene synthase from Penicillium roqueforti (PR-AS) has been probed with the farnesyl diphosphate analogues 6- and 14-fluoro farnesyl diphosphate (1a and 1c). Incubation of these analogues with PR-AS followed by analysis of the reaction products by GC-MS and NMR spectroscopy indicated that these synthetic FPP analogues were converted to the fluorinated germacrene A analogues 3b and 3c, respectively. In both cases the position of the fluorine atom prevented the formation of the eudesmane cation analogues 4b and 4c. These results highlight that germacrene A is an on-path reaction intermediate during PR-AS catalysis and shed light on the mechanism by which germacrene A is converted to eudesmane cation. They support the proposal that the role of PR-AS in the cyclisation is essentially passive in that it harnesses the inherent chemical reactivity present in the substrate by promoting the initial ionisation of farnesyl diphosphate and by acting as a productive template to steer the reaction through an effective series of cyclisations and rearrangements to (+)-aristolochene (7a). PMID:19225680

  8. Metal ion-binding properties of the diphosphate ester analogue, methylphosphonylphosphate, in aqueous solution.

    PubMed

    Song, B; Zhao, J; Gregán, F; Prónayová, N; Sajadi, S A; Sigel, H

    1999-01-01

    The stability constants of the 1:1 complexes formed between methylphosphonylphosphate (MePP(3-)), CH(3)P(O)(-) (2)-O-PO3(2-), and Mg(2+), Ca(2+), Sr(2+), Ba(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), or Cd(2+) (M(2+)) were determined by potentiometric pH titration in aqueous solution (25 degrees C; l = 0.1 M, NaNO(3)). Monoprotonated M(H;MePP) complexes play only a minor role. Based on previously established correlations for M(2+)-diphosphate monoester complex-stabilities and diphosphate monoester beta-group. basicities, it is shown that the M(Mepp)(-) complexes for Mg(2+) and the ions of the second half of the 3d series, including Zn(2+) and Cd(2+), are on average by about 0.15 log unit more stable than is expected based on the basicity of the terminal phosphate group in MePP(3-). In contrast, Ba(Mepp)(-) and Sr(Mepp)(-) are slightly less stable, whereas the stability for Ca(Mepp)(-) is as expected, based on the mentioned correlation. The indicated increased stabilities are explained by an increased basicity of the phosphonyl group compared to that of a phosphoryl one. For the complexes of the alkaline earth ions, especially for Ba(2+), it is suggested that outersphere complexation occurs to some extent. However, overall the M(Mepp)(-) complexes behave rather as expected for a diphosphate monoester ligand. PMID:18475908

  9. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    PubMed Central

    Wang, Jianrong; Li, Yangyuan; Liu, Danni

    2014-01-01

    Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos. PMID:25474088

  10. The distribution of carbonic anhydrase and ribulose diphosphate carboxylase in maize leaves.

    PubMed

    Poincelot, R P

    1972-09-01

    Extraction of maize (Zea mays) leaves by progressive grinding under suitably protective conditions yields total carbonic anhydrase activities (4800 units per milligram chlorophyll) comparable to the activity in spinach (Spinacia oleracea) leaves. The total ribulose diphosphate carboxylase activity was also equal to or greater than the best literature values for maize. Of the total leaf carbonic anhydrase, 72.5% on a chlorophyll basis was present in the mesophyll cells and 14.2% in the bundle-sheath cells. The distribution of the total leaf ribulose diphosphate carboxylase between the mesophyll and bundle-sheath cells was 42.0 and 48.7% respectively. There was three times as much total chlorophyll in extracts of the mesophyll cells compared with the bundle-sheath cells of maize. Similar results for the above distribution of the two enzymes were found using a differential grinding technique. The possible function of carbonic anhydrase in photosynthesis is discussed. The equal distribution of ribulose diphosphate carboxylase activity between the mesophyll and bundle-sheath cells casts doubt upon the hypothesis that a rigid biochemical compartmentation exists between these cell types in maize. PMID:16658170

  11. Metal Ion-Binding Properties of the Diphosphate Ester Analogue, Methylphosphonylphosphate, in Aqueous Solution

    PubMed Central

    Song, Bin; Zhao, Jing; Gregáň, Fridrich; Prónayová, Nadja; Sajadi, S. Ali A.

    1999-01-01

    The stability constants of the 1:1 complexes formed between methylphosphonylphosphate (MePP3-), CH3P(O)-2-O-PO32-, and Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, ​ or Cd2+ (M2+) were determined by potentiometric pH titration in aqueous solution (25 °C; l = 0.1 M, NaNO3). Monoprotonated M(H;MePP) complexes play only a minor role. Based on previously established correlations for M2+-diphosphate monoester complex-stabilities and diphosphate monoester β-group. basicities, it is shown that the M(Mepp)- complexes for Mg2+ and the ions of the second half of the 3d series, including Zn2+ and Cd2+, are on average by about 0.15 log unit more stable than is expected based on the basicity of the terminal phosphate group in MePP3-. In contrast, Ba(Mepp)- and Sr(Mepp)- are slightly less stable, whereas the stability for Ca(Mepp)- is as expected, based on the mentioned correlation. The indicated increased stabilities are explained by an increased basicity of the phosphonyl group compared to that of a phosphoryl one. For the complexes of the alkaline earth ions, especially for Ba2+, it is suggested that outersphere complexation occurs to some extent. However, overall the M(Mepp)- complexes behave rather as expected for a diphosphate monoester ligand. PMID:18475908

  12. Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

    SciTech Connect

    Liang, B.T.

    1989-06-01

    Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).

  13. Adenosine augments interleukin-10 production by microglial cells through an A2B adenosine receptor-mediated process

    PubMed Central

    Koscsó, Balázs; Csóka, Balázs; Selmeczy, Zsolt; Himer, Leonóra; Pacher, Pál; Virág, László; Haskó, György

    2011-01-01

    Microglia are activated by pathogen-associated molecular patterns and produce pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and is a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. Here, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of pro-inflammatory cytokines. Since the order of potency of selective AR agonists in inducing IL-10 production was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) ≥ 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS21680), and the A2BAR antagonist MRS-1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered shRNA blocked the enhancing effect of adenosine on IL-10 production confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia. PMID:22116830

  14. A(3) adenosine receptor ligands: history and perspectives.

    PubMed

    Baraldi, P G; Cacciari, B; Romagnoli, R; Merighi, S; Varani, K; Borea, P A; Spalluto, G

    2000-03-01

    Adenosine regulates many physiological functions through specific cell membrane receptors. On the basis of pharmacological studies and molecular cloning, four different adenosine receptors have been identified and classified as A(1), A(2A), A(2B), and A(3). These adenosine receptors are members of the G-protein-coupled receptor family. While adenosine A(1) and A(2A) receptor subtypes have been pharmacologically characterized through the use of selective ligands, the A(3) adenosine receptor subtype is presently under study in order to better understand its physio-pathological functions. Activation of adenosine A(3) receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase. Activation of A(3) adenosine receptors also causes the release of inflammatory mediators such as histamine from mast cells. These mediators are responsible for processes such as inflammation and hypotension. It has also been suggested that the A(3) receptor plays an important role in brain ischemia, immunosuppression, and bronchospasm in several animal models. Based on these results, highly selective A(3) adenosine receptor agonists and/or antagonists have been indicated as potential drugs for the treatment of asthma and inflammation, while highly selective agonists have been shown to possess cardioprotective effects. The updated material related to this field of research has been rationalized and arranged in order to offer an overview of the topic. PMID:10723024

  15. Comorbidities in Neurology: Is adenosine the common link?

    PubMed

    Boison, Detlev; Aronica, Eleonora

    2015-10-01

    Comorbidities in Neurology represent a major conceptual and therapeutic challenge. For example, temporal lobe epilepsy (TLE) is a syndrome comprised of epileptic seizures and comorbid symptoms including memory and psychiatric impairment, depression, and sleep dysfunction. Similarly, Alzheimer's disease (AD), Parkinson's disease (PD), and Amyotrophic Lateral Sclerosis (ALS) are accompanied by various degrees of memory dysfunction. Patients with AD have an increased likelihood for seizures, whereas all four conditions share certain aspects of psychosis, depression, and sleep dysfunction. This remarkable overlap suggests common pathophysiological mechanisms, which include synaptic dysfunction and synaptotoxicity, as well as glial activation and astrogliosis. Astrogliosis is linked to synapse function via the tripartite synapse, but astrocytes also control the availability of gliotransmitters and adenosine. Here we will specifically focus on the 'adenosine hypothesis of comorbidities' implying that astrocyte activation, via overexpression of adenosine kinase (ADK), induces a deficiency in the homeostatic tone of adenosine. We present evidence from patient-derived samples showing astrogliosis and overexpression of ADK as common pathological hallmark of epilepsy, AD, PD, and ALS. We discuss a transgenic 'comorbidity model', in which brain-wide overexpression of ADK and resulting adenosine deficiency produces a comorbid spectrum of seizures, altered dopaminergic function, attentional impairment, and deficits in cognitive domains and sleep regulation. We conclude that dysfunction of adenosine signaling is common in neurological conditions, that adenosine dysfunction can explain co-morbid phenotypes, and that therapeutic adenosine augmentation might be effective for the treatment of comorbid symptoms in multiple neurological conditions. PMID:25979489

  16. Adenosine: Essential for life but licensed to kill

    PubMed Central

    Gama, Vivian; Deshmukh, Mohanish

    2016-01-01

    In this issue of Molecular Cell, Long et al. (Long et al., 2013) report a cell death priming mechanism activated by p53 that senses extracellular adenosine accumulated following chemotherapy or hypoxia, providing a novel connection between adenosine signaling and apoptosis. PMID:25884366

  17. Targeting of Adenosine Receptors in Ischemia-Reperfusion Injury

    PubMed Central

    Laubach, Victor E.; French, Brent A.; Okusa, Mark D.

    2010-01-01

    Importance of the field Ischemia-reperfusion (IR) injury is a common clinical problem after transplantation as well as myocardial infarction and stroke. IR initiates an inflammatory response leading to rapid tissue damage. Adenosine, produced in response to IR, is generally considered as a protective signaling molecule and elicits its physiological responses through four distinct adenosine receptors. The short half-life, lack of specificity, and rapid metabolism limits the use of adenosine as a therapeutic agent. Thus intense research efforts have focused on the synthesis and implementation of specific adenosine receptor agonists and antagonists as potential therapeutic agents for a variety of inflammatory conditions including IR injury. Areas covered by this review This review summarizes current knowledge on IR injury with a focus on lung, heart, and kidney, and examines studies that have advanced our understanding of the role of adenosine receptors and the therapeutic potential of adenosine receptor agonists and antagonists for the prevention of IR injury. What the reader will gain The reader will gain insight into the role of adenosine receptor signaling in IR injury. Take home message No clinical therapies are currently available that specifically target IR injury; however, targeting of specific adenosine receptors may offer therapeutic strategies in this regard. PMID:21110787

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

    PubMed

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

    2015-05-01

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

  19. Adenosine receptors and asthma in humans.

    PubMed

    Wilson, C N

    2008-10-01

    According to an executive summary of the GINA dissemination committee report, it is now estimated that approximately 300 million people (5% of the global population or 1 in 20 persons) have asthma. Despite the scientific progress made over the past several decades toward improving our understanding of the pathophysiology of asthma, there is still a great need for improved therapies, particularly oral therapies that enhance patient compliance and that target new mechanisms of action. Adenosine is an important signalling molecule in human asthma. By acting on extracellular G-protein-coupled ARs on a number of different cell types important in the pathophysiology of human asthma, adenosine affects bronchial reactivity, inflammation and airway remodelling. Four AR subtypes (A(1), A(2a), A(2b) and A(3)) have been cloned in humans, are expressed in the lung, and are all targets for drug development for human asthma. This review summarizes what is known about these AR subtypes and their function in human asthma as well as the pros and cons of therapeutic approaches to these AR targets. A number of molecules with high affinity and high selectivity for the human AR subtypes have entered clinical trials or are poised to enter clinical trials as anti-asthma treatments. With the availability of these molecules for testing in humans, the function of ARs in human asthma, as well as the safety and efficacy of approaches to the different AR targets, can now be determined. PMID:18852693

  20. The Role of Adenosine Signaling in Sickle Cell Therapeutics

    PubMed Central

    Field, Joshua J.; Nathan, David G.; Linden, Joel

    2014-01-01

    Recent data suggest a role for adenosine signaling in the pathogenesis of sickle cell disease (SCD). Signaling through the adenosine A2A receptor (A2AR) has demonstrated beneficial effects in SCD. Activation of A2ARs decreases inflammation in mice and patients with SCD largely by blocking activation of invariant NKT cells. Decreased inflammation may reduce the severity of vaso-occlusive crises. In contrast, adenosine signaling through the A2B receptor (A2BR) may be detrimental for patients with SCD. Priapism and the formation of sickle erythrocytes may be a consequence of A2BR activation on corpus cavernosal cells and erythrocytes, respectively. Whether adenosine signaling predominantly occurs through A2ARs or A2BRs may depend on differing levels of adenosine and disease state (steady state versus crisis). There may be opportunities to develop novel therapeutic approaches targeting A2ARs and/or A2BRs for patients with SCD. PMID:24589267

  1. Chronic benzodiazepine treatment and cortical responses to adenosine and GABA.

    PubMed

    Mally, J; Connick, J H; Stone, T W

    1990-10-22

    The effects of chronic treatment of mice with clonazepam have been examined on the responses of neocortical slices to adenosine, 5-hydroxytryptamine (5-HT) and gamma-aminobutyric acid (GABA). Responses to these agonists were measured as changes in the depolarisation induced by N-methyl-D-aspartate (NMDA). Added to the superfusion medium diazepam blocked responses to adenosine but not 5-HT; this effect was not observed with 2-chloroadenosine or in the presence of 2-hydroxynitrobenzylthioguanosine. GABA was inactive in control slices but chronic treatment with clonazepam induced responses to GABA and enhanced responses to adenosine but not 5-HT. It is suggested that the induction of GABA responses may reflect the up-regulation of GABA receptors, but the increase of adenosine responses by clonazepam implies that there is no simple relationship between adenosine receptor binding and functional responses. PMID:1979931

  2. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  3. An adenosine kinase inhibitor, ABT-702, inhibits spinal nociceptive transmission by adenosine release via equilibrative nucleoside transporters in rat.

    PubMed

    Otsuguro, Ken-ichi; Tomonari, Yuki; Otsuka, Saori; Yamaguchi, Soichiro; Kon, Yasuhiro; Ito, Shigeo

    2015-10-01

    Adenosine kinase (AK) inhibitor is a potential candidate for controlling pain, but some AK inhibitors have problems of adverse effects such as motor impairment. ABT-702, a non-nucleoside AK inhibitor, shows analgesic effect in animal models of pain. Here, we investigated the effects of ABT-702 on synaptic transmission via nociceptive and motor reflex pathways in the isolated spinal cord of neonatal rats. The release of adenosine from the spinal cord was measured by HPLC. ABT-702 inhibited slow ventral root potentials (sVRPs) in the nociceptive pathway more potently than monosynaptic reflex potentials (MSRs) in the motor reflex pathway. The inhibitory effects of ABT-702 were mimicked by exogenously applied adenosine, blocked by 8CPT (8-cyclopentyl-1,3-dipropylxanthine), an adenosine A1 receptor antagonist, and augmented by EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine), an adenosine deaminase (ADA) inhibitor. Equilibrative nucleoside transporter (ENT) inhibitors reversed the effects of ABT-702, but not those of adenosine. ABT-702 released adenosine from the spinal cord, an effect that was also reversed by ENT inhibitors. The ABT-702-facilitated release of adenosine by way of ENTs inhibits nociceptive pathways more potently than motor reflex pathways in the spinal cord via activation of A1 receptors. This feature is expected to lead to good analgesic effects, but, caution may be required for the use of AK inhibitors in the case of ADA dysfunction or a combination with ENT inhibitors. PMID:26066576

  4. Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains.

    PubMed

    Lansdon, Eric B; Fisher, Andrew J; Segel, Irwin H

    2004-04-13

    first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1. PMID:15065880

  5. Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

    PubMed

    Kusek, Justyna; Yang, Qiong; Witek, Martin; Gruber, Christian W; Nanoff, Christian; Freissmuth, Michael

    2015-01-01

    Cell-permeable orthosteric ligands can assist folding of G protein-coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress. PMID:25354767

  6. Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+.

    PubMed

    Wu, D; Boyer, P D

    1986-06-01

    When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions. PMID:2873834

  7. Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

    PubMed Central

    Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

    2014-01-01

    Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

  8. Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus.

    PubMed

    Zhang, Dali; Xiong, Wei; Jackson, Michael F; Parkinson, Fiona E

    2016-07-01

    Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5'-nucleotidase activity. Wild-type (CD73(+/+)) and ecto-5'-nucleotidase-deficient (CD73(-/-)) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73(+/+) mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73(+/+) mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg(2+) conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5'-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73(-/-) mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine A1 receptors in hippocampus, mediated, at least in part, via reduced ecto-5'-nucleotidase activity. PMID:27189965

  9. Identification of possible adenosine receptors in vascular smooth muscle

    SciTech Connect

    Doctrow, S.R.

    1985-01-01

    Adenosine is a vasodilator and has been implicated in increased blood flow in tissues that undergo energy deficiency. During conditions such as hypoxia and ischemia, adenosine is produced and is said to increase blood flow by relaxing the vascular smooth muscle (VSM) lining the resistance vessels. The goal of this research was to identify receptors that might be responsible for adenosine-mediated VSM relaxation. When an insoluble fraction from calf aortic VSM was incubated with /sup 32/P-ATP, two components were phosphorylated. One was identified as myosin light chain by MW, pl, and immunoprecipitation. The other product was identified as phosphatidylinositol-4-phosphate (DPI) by tic. Both phosphorylations were inhibited by adenosine and by 5'-chloro-5'-deoxyadenosine (Cl-Ado). DPI production was much more sensitive to the nucleosides than was myosin phosphorylation. Neither inhibition involved change in cAMP production. Phosphatidylinositol (Pl) kinase in the VSM membranes required magnesium, was activated and solubilized by Triton X-100, and phosphorylated both endogenous and exogenous Pl. Cl-Ado inhibited Pl kinase in a manner competitive with respect to ATP and noncompetitive with respect to Pl. Adenosine and adenosine analogs modified in the ribose ring were inhibitors with potencies comparable to that of Cl-Ado. Adenine nucleotides and purine-modified adenosine analogs were weaker inhibitors than Cl-Ado.

  10. Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus

    PubMed Central

    Zhang, Dali; Xiong, Wei; Jackson, Michael F.

    2016-01-01

    Ethanol has many pharmacological effects, including increases in endogenous adenosine levels and adenosine receptor activity in brain. Ethanol consumption is associated with both positive and negative health outcomes, but tolerance to the behavioral effects of ethanol can lead to increased consumption, which increases the risk of negative health outcomes. The present study was performed to test whether a 7-day treatment with ethanol is linked to reduced adenosine signaling and whether this is a consequence of reduced ecto-5′-nucleotidase activity. Wild-type (CD73+/+) and ecto-5′-nucleotidase-deficient (CD73−/−) mice were treated with ethanol (2 g/kg) or saline for 7 days. In CD73+/+ mice, repeated ethanol treatment reduced the hypothermic and ataxic effects of acute ethanol, indicating the development of tolerance to the acute effects of ethanol. In CD73+/+ mice, this 7-day ethanol treatment led to increased hippocampal synaptic activity and reduced adenosine A1 receptor activity under both basal and low Mg2+ conditions. These effects of ethanol tolerance were associated with an 18% decrease in activity of ecto-5′-nucleotidase activity in hippocampal cell membranes. In contrast, ethanol treatment was not associated with changes in synaptic activity or adenosine signaling in hippocampus from CD73−/− mice. These data indicate that ethanol treatment is associated with a reduction in adenosine signaling through adenosine A1 receptors in hippocampus, mediated, at least in part, via reduced ecto-5′-nucleotidase activity. PMID:27189965

  11. Characteristic molecular vibrations of adenosine receptor ligands.

    PubMed

    Chee, Hyun Keun; Yang, Jin-San; Joung, Je-Gun; Zhang, Byoung-Tak; Oh, S June

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. PMID:25622891

  12. Increased Cortical Extracellular Adenosine Correlates with Seizure Termination

    PubMed Central

    Van Gompel, Jamie J.; Bower, Mark R.; Worrell, Gregory A.; Stead, Matt; Chang, Su-Youne; Goerss, Stephan J.; Kim, Inyong; Bennet, Kevin E.; Meyer, Fredric B.; Marsh, W. Richard; Blaha, Charles D.; Lee, Kendall H.

    2014-01-01

    Objective Seizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent upon neurotransmitters of which little is known regarding their peri-ictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Further, microdialysis studies in humans suggest adenosine is elevated peri-ictally, but the relationship to the seizure is obscured by its temporal measurement limitations. Because electrochemical techniques can provide vastly superior temporal resolution, we test the hypothesis that extracellular adenosine concentrations rise during seizure termination in an animal model and humans using electrochemistry. Methods White farm swine (n=45) were used in an acute cortical model of epilepsy and 10 human epilepsy patients were studied during intraoperative electrocorticography (Ecog). Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS) based fast scan cyclic voltametry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine specific triangular waveform or biosensors respectively. Results Simultaneous Ecog and electrochemistry demonstrated an average adenosine rise of 260% compared to baseline at 7.5 ± 16.9 seconds with amperometry (n=75 events) and 2.6 ± 11.2 seconds with FSCV (n=15 events) prior to electrographic seizure termination. In agreement with these animal data, adenosine elevation prior to seizure termination in a human patient utilizing FSCV was also seen. Significance Simultaneous Ecog and electrochemical recording supports the hypothesis that adenosine rises prior to seizure termination, suggesting that adenosine itself may be responsible for seizure termination. Future work using intraoperative WINCS based FSCV recording may help to elucidate the precise relationship between adenosine and seizure termination. PMID:24483230

  13. Why do asthmatic subjects respond so strongly to inhaled adenosine?

    PubMed

    Meade, C J; Dumont, I; Worrall, L

    2001-08-01

    Bronchospasm induced by adenosine is blocked by representatives of all the major classes of drugs used in the treatment of asthma. Understanding the mechanism of this bronchospasm may help understand the way these drugs work. Clinical studies have suggested involvement of neural pathways, mast-like cells and mediators such as histamine, serotonin and lipoxygenase products. There is a strong link between responsiveness to adenosine and eosinophilia. In different animal models A1, A2b and A3 adenosine receptor subclasses have all been implicated in inducing bronchospasm. whilst occupation of the A2a receptor generally has no, or the opposite effect. At least two different mechanisms, both involving neural pathways, exist. One, involving the adenosine A1 receptor, functions in mast cell depleted animals; the other requires interaction with a population of mast-like cells activated over A2b or A3 receptors. Not only histamine but also serotonin and lipoxygenase products released from the mast-like cells are potential mediators. In animal models good reactivity to adenosine receptor agonists is generally only found when the animals are first sensitized and exposed to allergen in ways likely to induce an allergic inflammation. An exception is the BDE rat, which reacts to adenosine receptor agonists such as APNEA or NECA even without allergen exposure. This rat strain does however show evidence of spontaneous eosinophilic inflammation in the lung even without immunization. As mast cells both release adenosine and respond to adenosine, adenosine provides a non-specific method of amplifying specific signals resulting from IgE/antigen interaction. This mechanism may not only have a pathological significance in asthma; it may be part of a normal bodily defense response that in asthmatic subjects is inappropriately activated. PMID:11521747

  14. Adenosine reversal of in vivo hepatic responsiveness to insulin.

    PubMed

    McLane, M P; Black, P R; Law, W R; Raymond, R M

    1990-01-01

    Modulation by adenosine of hepatic responsiveness to insulin was investigated in vivo in 10 healthy mongrel dogs of both sexes by determining net hepatic glucose output (NHGO) in response to insulin during the presence or absence of exogenous adenosine infusion. In addition, two separate series of experiments were performed to study the effect of adenosine (n = 7) or glucagon (n = 5) on NHGO. Basal NHGO, quantitated via the Fick principle, was significantly decreased by insulin infusion (4 U/min; 4.8 +/- 0.6 vs. -1.7 +/- 2.6 mg.kg-1.min-1, P less than 0.05). The addition of an intrahepatic arterial infusion of adenosine (10 mumol/min) during insulin infusion caused glucose output to return to basal levels (insulin, -1.7 +/- 2.6 mg.kg-1.min-1; insulin + adenosine, 3.8 +/- 1.6 mg.kg-1.min-1, P less than 0.05). The addition of intrahepatic arterial saline (control) during insulin infusion had no effect on insulin's action (insulin, -1.0 +/- 1.9 mg.kg-1.min-1; insulin + saline, -1.2 +/- 1.6 mg.kg-1.min-1, P greater than 0.05). Hepatic glucose, lactate, and oxygen deliveries were not affected during either insulin or insulin plus adenosine infusion. Intrahepatic arterial infusion of adenosine alone had no effect on NHGO, whereas intrahepatic arterial infusion of glucagon alone stimulated glucose output approximately fivefold (basal, 2.7 +/- 0.4 mg.kg-1.min-1; glucagon, 15.5 +/- 1.2 mg.kg-1.min-1, P less than 0.01). These results show that adenosine completely reversed the inhibition by insulin of NHGO. These data suggest that adenosine may act as a modulator of insulin action on the liver. PMID:2210062

  15. Adenosine deaminase in disorders of purine metabolism and in immune deficiency

    SciTech Connect

    Tritsch, G.L.

    1985-01-01

    This book consists of five parts and a section of poster papers. Some of the selection titles are: Adenosine Deaminase Impairment and Ribonucleotide Reductase in Human Cells; Adenosine Deaminase and Malignant Cells; Inhibition of Adenosine Deaminase to Increase the Antitumor Activity of Adenine Nucleoside Analogues; and Molecular Biology of the Adenosine Deaminase Gene and Messenger RNA.

  16. Structure of geranyl diphosphate C-methyltransferase from Streptomyces coelicolor and implications for the mechanism of isoprenoid modification†

    PubMed Central

    Köksal, Mustafa; Chou, Wayne K. W.; Cane, David E.; Christianson, David W.

    2012-01-01

    Geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a non-canonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl-S-thiolodiphosphate (GSPP) along with S-adenosyl-l-homocysteine in the cofactor binding site, resulting from in situ demethylation of S-adenosyl-l-methionine, at 2.05 Å and 1.82 Å resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. S-adenosyl-l-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor S-adenosyl-l-methionine. These structures provide important clues regarding the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation-π interactions. PMID:22455498

  17. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle

    PubMed Central

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G.; Köllner, Tobias G.

    2016-01-01

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

  18. Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

    PubMed

    Beran, Franziska; Rahfeld, Peter; Luck, Katrin; Nagel, Raimund; Vogel, Heiko; Wielsch, Natalie; Irmisch, Sandra; Ramasamy, Srinivasan; Gershenzon, Jonathan; Heckel, David G; Köllner, Tobias G

    2016-03-15

    Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors. PMID:26936952

  19. Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

    SciTech Connect

    Flentke, G.R.; Frey, P.A. )

    1990-03-06

    UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5{prime}-diphosphate chloroacetol (UDC) and uridine 5{prime}-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K{sub D} of 0.110 mM and k{sub inact} of 0.84 min{sup {minus}1} at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD{sup +}. The inactivation of epimerase by uridine 5{prime}-diphosphate ({sup 2}H{sub 2})chloroacetol proceeds with a primary kinetic isotope effect (k{sub H}/k{sub D}) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD{sup +} at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD{sup +} is proposed to be the chromophore with {lambda}{sub max} at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction.

  20. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    SciTech Connect

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  1. Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling

    PubMed Central

    Mi, Tiejuan; Abbasi, Shahrzad; Zhang, Hong; Uray, Karen; Chunn, Janci L.; Xia, Ling Wei; Molina, Jose G.; Weisbrodt, Norman W.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2008-01-01

    Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor–mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine–induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada–/– and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder. PMID:18340377

  2. Impact of protease inhibitors on intracellular concentration of tenofovir-diphosphate among HIV-1 infected patients

    PubMed Central

    Lahiri, Cecile D.; Tao, Sijia; Jiang, Yong; Sheth, Anandi N.; Acosta, Edward P.; Marconi, Vincent C.; Armstrong, Wendy S.; Schinazi, Raymond F.; Vunnava, Aswani; Sanford, Sara; Ofotokun, Ighovwerha

    2015-01-01

    Intracellular nucleoside reverse transcriptase inhibitor (NRTI) concentrations are associated with plasma HIV-1 response. Coadministration of protease inhibitors with NRTIs can affect intra-cellular concentrations due to protease inhibitor inhibition of efflux transporters. Tenofovir-diphosphate (TFV-DP) concentrations within peripheral blood mononuclear cells were compared among individuals receiving either atazanavir or darunavir-based regimens. There was a trend towards higher TFV-DP concentrations among women and among participants receiving atazanavir. TFV-DP intracellular concentrations were positively associated with undetectable plasma HIV-1 RNA. PMID:25870991

  3. Strength Characteristics of Resorbable Osteoconductive Ceramics Based on Diphosphates of Calcium and Alkali Metals

    NASA Astrophysics Data System (ADS)

    Putlayev, V. I.; Evdokimov, P. V.; Garshev, A. V.; Prosvirin, D. V.; Klimashina, E. S.; Safronova, T. V.; Ivanov, V. K.

    2014-02-01

    An investigation into the strength characteristics of ceramics based on diphosphates Ca(3- x)М2 x (PO4)2 ( x = 0-1 and М = Na, K) provides evidence of composition strengthening in the range х = 0.6-0.8 containing the greatest amount of the supercooled high-temperature modification α-СаМРО4. The method of high-temperature x-ray diffractometry is used to examine thermal expansion of rhenanite phases of СаМРО4.

  4. The Synthesis of Guanosine 5′-Diphosphate l-Fucose from Guanosine 5′-Diphosphate 3,5-d-[3H]Mannose Catalyzed by an Enzyme Extract from Fruits of the Flax

    PubMed Central

    Barber, George A.

    1980-01-01

    An enzyme system from fruits of the flax plant is described that catalyzes the synthesis of the sugar nucleotide guanosine 5′-diphosphate l-fucose from guanosine 5′-diphosphate d-mannose with the intermediate formation of guanosine 5′-diphosphate 4-keto-6-deoxy-d-mannose. Tritium from-[3H]H2O was incorporated into the l-fucose portion of the sugar nucleotide in the course of the reaction, and tritium at the 3,5-carbons of the d-mannose moiety of GDP-d-mannose was exchanged with protons in the medium. These results support a mechanism of synthesis analogous to that proposed for the formation of l-rhamnose and other 6-deoxy sugars. PMID:16661431

  5. Impact of the bioresorbable vascular scaffold surface area on on-treatment platelet reactivity.

    PubMed

    Gross, Lisa; Sibbing, Dirk; Eickhoff, Madeleine; Baquet, Moritz; Orban, Martin; Krieg, Anne; Grujic, Katarina; Theiss, Hans D; Brunner, Stefan; Teupser, Daniel; Holdt, Lesca; Massberg, Steffen; Mehilli, Julinda

    2016-07-01

    While promising data with the novel bioresorbable vascular scaffold (BVS) are accumulating, signals of scaffold thrombosis (ST) were noted in recent reports. We aimed to assess the relationship between the total surface area (TSA) of implanted everolimus-eluting BVSs and the on-treatment adenosine diphosphate (ADP)-induced platelet reactivity in patients undergoing percutaneous coronary intervention (PCI). 202 consecutive patients undergoing BVS implantation and platelet function testing were included. For investigating the impact of the scaffold surface on platelet reactivity, patients were stratified into two groups regarding the median BVS TSA. The on-treatment ADP-induced platelet reactivity was determined with the Multiplate analyzer and 30-day follow-up was available in 98% of patients. ADP-induced platelet aggregation values (median, [IQR]) did not differ between the two study groups (12.0 [9.0-19.0] U for patients with TSA > 1.39 cm(2) and 13.0 [9.0-19.5] U for patients with TSA ≤ 1.39 cm(2); p = 0.69). No correlation was observed between the BVS TSA and levels of platelet reactivity (Spearman rank correlation = -0.10, p = 0.16). At 30 days after PCI, two early STs (1%) were documented. Thus, in patients on a dual antiplatelet treatment regimen following BVS implantation, the extent of blood-to-BVS contact surface does not negatively affect levels of on-treatment platelet reactivity. PMID:26940818

  6. Extracellular Adenosine Mediates a Systemic Metabolic Switch during Immune Response

    PubMed Central

    Bajgar, Adam; Kucerova, Katerina; Jonatova, Lucie; Tomcala, Ales; Schneedorferova, Ivana; Okrouhlik, Jan; Dolezal, Tomas

    2015-01-01

    Immune defense is energetically costly, and thus an effective response requires metabolic adaptation of the organism to reallocate energy from storage, growth, and development towards the immune system. We employ the natural infection of Drosophila with a parasitoid wasp to study energy regulation during immune response. To combat the invasion, the host must produce specialized immune cells (lamellocytes) that destroy the parasitoid egg. We show that a significant portion of nutrients are allocated to differentiating lamellocytes when they would otherwise be used for development. This systemic metabolic switch is mediated by extracellular adenosine released from immune cells. The switch is crucial for an effective immune response. Preventing adenosine transport from immune cells or blocking adenosine receptor precludes the metabolic switch and the deceleration of development, dramatically reducing host resistance. Adenosine thus serves as a signal that the “selfish” immune cells send during infection to secure more energy at the expense of other tissues. PMID:25915062

  7. Alterations of adenosine A1 receptors in morphine dependence.

    PubMed

    Kaplan, G B; Leite-Morris, K A; Sears, M T

    1994-09-19

    The possibility that central adenosine A1 and A2a receptors mediate opiate dependence was examined in morphine-treated mice using radioligand binding methods. Mice treated with morphine for 72 h demonstrated significant increases in naloxone precipitated abstinence behaviors of jumping, wet-dog shakes, teeth chattering, forepaw trends, forepaw tremors and diarrhea compared to vehicle-treated mice. Increased concentrations of cortical adenosine A1 receptor sites, but not striatal adenosine A2a sites, were found in saturation binding studies from morphine-dependent mice. Decreases in cortical A1 agonist binding affinity values along with increases in agonist binding sites were demonstrated in competition binding studies. These results suggest that adaptive changes of upregulation and sensitization of adenosine A1 receptors play a role in mediating the opiate abstinence syndrome. PMID:7820640

  8. Oritavancin Diphosphate

    PubMed Central

    Cada, Dennis J.; Baker, Danial E.

    2014-01-01

    Each month, subscribers to The Formulary Monograph Service receive 5 to 6 well-documented monographs on drugs that are newly released or are in late phase 3 trials. The monographs are targeted to Pharmacy & Therapeutics Committees. Subscribers also receive monthly 1-page summary monographs on agents that are useful for agendas and pharmacy/nursing in-services. A comprehensive target drug utilization evaluation/medication use evaluation (DUE/MUE) is also provided each month. With a subscription, the monographs are sent in print and are also available on-line. Monographs can be customized to meet the needs of a facility. A drug class review is now published monthly with The Formulary Monograph Service. Through the cooperation of The Formulary, Hospital Pharmacy publishes selected reviews in this column. For more information about The Formulary Monograph Service, call The Formulary at 800-322-4349. The December 2014 monograph topics are olodaterol, peginterferon beta-1a, testosterone nasal gel, ferric citrate corredination complex, and safinamide. The Safety MUE is on olodaterol. PMID:25673895

  9. Oritavancin diphosphate.

    PubMed

    Cada, Dennis J; Baker, Danial E

    2014-12-01

    Each month, subscribers to The Formulary Monograph Service receive 5 to 6 well-documented monographs on drugs that are newly released or are in late phase 3 trials. The monographs are targeted to Pharmacy & Therapeutics Committees. Subscribers also receive monthly 1-page summary monographs on agents that are useful for agendas and pharmacy/nursing in-services. A comprehensive target drug utilization evaluation/medication use evaluation (DUE/MUE) is also provided each month. With a subscription, the monographs are sent in print and are also available on-line. Monographs can be customized to meet the needs of a facility. A drug class review is now published monthly with The Formulary Monograph Service. Through the cooperation of The Formulary, Hospital Pharmacy publishes selected reviews in this column. For more information about The Formulary Monograph Service, call The Formulary at 800-322-4349. The December 2014 monograph topics are olodaterol, peginterferon beta-1a, testosterone nasal gel, ferric citrate corredination complex, and safinamide. The Safety MUE is on olodaterol. PMID:25673895

  10. Proton transfer in oxidized adenosine self-aggregates.

    PubMed

    Capobianco, Amedeo; Caruso, Tonino; Celentano, Maurizio; La Rocca, Mario Vincenzo; Peluso, Andrea

    2013-10-14

    The UV-vis and the IR spectra of derivativized adenosine in dichloromethane have been recorded during potentiostatic oxidation at an optically transparent thin layer electrode. Oxidized adenosine shows a broad Zundel like absorption extending from 2800 up to 3600 cm(-1), indicating that a proton transfer process is occurring. Theoretical computations predict that proton transfer is indeed favored in oxidized 1:1 self-association complexes and allow to assign all the observed transient spectroscopic signals. PMID:24116647

  11. The A3 adenosine receptor: history and perspectives.

    PubMed

    Borea, Pier Andrea; Varani, Katia; Vincenzi, Fabrizio; Baraldi, Pier Giovanni; Tabrizi, Mojgan Aghazadeh; Merighi, Stefania; Gessi, Stefania

    2015-01-01

    By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health. PMID:25387804

  12. Myocardial perfusion scintigraphy during maximal coronary artery vasodilation with adenosine

    SciTech Connect

    Verani, M.S.; Mahmarian, J.J. )

    1991-05-21

    Pharmacologic coronary vasodilation as an adjunct to thallium-201 myocardial perfusion scintigraphy provides an important alternative form of stress that has been increasingly used in patients unable to perform an exercise stress test. Although dipyridamole has traditionally been used for this purpose, there are several compelling reasons why adenosine may be a preferable agent. First, dipyridamole acts by blocking the reuptake and transport of adenosine, which is the effective substance responsible for coronary vasodilation. Second, exogenous adenosine has a very short half-life (less than 2 seconds), which explains its very short duration of action as well as the brief, self-limiting duration of its side effects. Third, the adenosine infusion is controllable and may be increased or decreased as desired. Fourth, the coronary vasodilation induced by the doses of adenosine we recommend (140 micrograms/kg/min) may be more profound than that induced by the standard dipyridamole dose. Our experience to date, with nearly 1,000 patients studied, shows the adenosine thallium-201 test to be practical and well tolerated, with high sensitivity (87%) and specificity (94%) for detecting coronary artery disease.

  13. Detrimental effects of adenosine signaling in sickle cell disease

    PubMed Central

    Zhang, Yujin; Dai, Yingbo; Wen, Jiaming; Zhang, Weiru; Grenz, Almut; Sun, Hong; Tao, Lijian; Lu, Guangxiu; Alexander, Danny C; Milburn, Michael V; Carter-Dawson, Louvenia; Lewis, Dorothy E; Zhang, Wenzheng; Eltzschig, Holger K; Kellems, Rodney E; Blackburn, Michael R; Juneja, Harinder S; Xia, Yang

    2016-01-01

    Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A2B receptor (A2BR)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A2BR has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease. PMID:21170046

  14. Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation.

    PubMed

    Hucul, J A; Henshaw, E C; Young, D A

    1985-12-15

    A sensitive assay method developed to examine the effects of subtle, physiologically relevant, changes in the levels of adenine and guanine mono-, di-, and triphosphorylated nucleotides specifically on the initiation of protein synthesis is described. Initiation rates are quantified by measuring the amount of protein synthesis resulting from the run-off of ribosomes which have initiated during defined intervals in a modified in vitro protein-synthesizing system developed from Ehrlich ascites tumor cell lysates (Henshaw, E.C., and Panniers, R. (1983) Methods Enzymol. 101, 616-629). The modifications include the attenuation of the ATP-regenerating system so that the relative nucleotide levels more nearly reflect actual intracellular conditions. With this system the rate of initiation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios, but indifferent to the absolute levels of either diphosphate. While the tight coupling of these two ratios by endogenous nucleoside diphosphate kinase activity prevents the independent manipulation of either ratio, the data do eliminate both AMP and GMP per se as inhibitory species. The close agreement of our data calculated in terms of energy charge to previously published results on overall rates of protein synthesis in rat thymocytes (Mendelsohn, S.K., Nordeen, S.K., and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79, 53-60) continues to suggest a physiologically relevant regulatory influence of subtle changes in nucleotides acting at the level of the initiation reaction. PMID:2999123

  15. Nucleoside diphosphate kinase of Saccharomyces cerevisiae, Ynk1p: localization to the mitochondrial intermembrane space.

    PubMed Central

    Amutha, Boominathan; Pain, Debkumar

    2003-01-01

    Nucleoside diphosphate kinase (NDPK) is a highly conserved multifunctional enzyme. It catalyses the transfer of gamma phosphates from nucleoside triphosphates to nucleoside diphosphates by a mechanism that involves formation of an autophosphorylated enzyme intermediate. The phosphate is usually supplied by ATP. NDPK activity in different subcellular compartments may regulate the crucial balance between ATP and GTP or other nucleoside triphosphates. NDPKs are homo-oligomeric proteins and are predominantly localized in the cytosol. In this paper, we demonstrate that in Saccharomyces cerevisiae a small fraction of total NDPK activity encoded by YNK1 is present in the intermembrane space (IMS) of mitochondria, and the corresponding protein Ynk1p in the IMS represents approx. 0.005% of total mitochondrial proteins. Ynk1p, synthesized as a single gene product, must therefore be partitioned between cytoplasm and mitochondrial IMS fractions. A mechanism for this partitioning is suggested by our observations that interaction with a 40 kDa protein of the translocase of outer mitochondrial membrane (Tom40p), occurs preferentially with unfolded, unphosphorylated forms of Ynk1p. A population of newly translated, but not yet folded or autophosphorylated, Ynk1p intermediates may be imported into the IMS of mitochondria and trapped there by subsequent folding and oligomerization. Within the small volume of the IMS, Ynk1p may be more concentrated and may be required to supply GTP to several important proteins in this compartment. PMID:12472466

  16. A cesium copper vanadyl-diphosphate: Synthesis, crystal structure and physical properties

    NASA Astrophysics Data System (ADS)

    Shvanskaya, Larisa; Yakubovich, Olga; Bychkov, Andrey; Shcherbakov, Vasiliy; Golovanov, Alexey; Zvereva, Elena; Volkova, Olga; Vasiliev, Alexander

    2015-02-01

    A non-centrosymmetric orthorhombic diphosphate, Cs2Cu1+x(VO)2-x(P2O7)2 (x=0.1) with a=13.7364(2) Å, b=9.2666(2) Å, c=11.5678(2) Å, Z=4, has been isolated. Its 3D framework is built from Cu atoms in square pyramidal and square planar coordination, VO5 tetragonal pyramids and P2O7 diphosphate groups, sharing vertices. Large channels are fulfilled by cesium atoms. The ESR study reveals a similarity in behaviour of two paramagnetic (Cu and V) subsystems. The temperature dependences of the ESR linewidth and static magnetic susceptibility data present evidences for a cluster type magnetic ordering in the title compound at T*=22 K. The weakness of the relevant anomalies reflects presumably obvious Cu2+ ions and (VO)2+ units disorder in the system. It is supposed that the charge and geometry of the framework are controlled by the Cu2+/(VO)2+ ratio; its variation may lead to a design of new materials.

  17. Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria: Three Steps Catalyzed by Three Enzymes

    PubMed Central

    2015-01-01

    Squalene (SQ) is an intermediate in the biosynthesis of sterols in eukaryotes and a few bacteria and of hopanoids in bacteria where they promote membrane stability and the formation of lipid rafts in their hosts. The genes for hopanoid biosynthesis are typically located on clusters that consist of four highly conserved genes—hpnC, hpnD, hpnE, and hpnF—for conversion of farnesyl diphosphate (FPP) to hopene or related pentacyclic metabolites. While hpnF is known to encode a squalene cyclase, the functions for hpnC, hpnD, and hpnE are not rigorously established. The hpnC, hpnD, and hpnE genes from Zymomonas mobilis and Rhodopseudomonas palustris were cloned into Escherichia coli, a bacterium that does not contain genes homologous to hpnC, hpnD, and hpnE, and their functions were established in vitro and in vivo. HpnD catalyzes formation of presqualene diphosphate (PSPP) from two molecules of FPP; HpnC converts PSPP to hydroxysqualene (HSQ); and HpnE, a member of the amine oxidoreductase family, reduces HSQ to SQ. Collectively the reactions catalyzed by these three enzymes constitute a new pathway for biosynthesis of SQ in bacteria. PMID:26258173

  18. Geranylgeranyl Diphosphate Synthase Modulates Fetal Lung Branching Morphogenesis Possibly through Controlling K-Ras Prenylation.

    PubMed

    Jia, Wen-Jun; Jiang, Shan; Tang, Qiao-Li; Shen, Di; Xue, Bin; Ning, Wen; Li, Chao-Jun

    2016-06-01

    G proteins play essential roles in regulating fetal lung development, and any defects in their expression or function (eg, activation or posttranslational modification) can lead to lung developmental malformation. Geranylgeranyl diphosphate synthase (GGPPS) can modulate protein prenylation that is required for protein membrane-anchoring and activation. Here, we report that GGPPS regulates fetal lung branching morphogenesis possibly through controlling K-Ras prenylation during fetal lung development. GGPPS was continuously expressed in lung epithelium throughout whole fetal lung development. Specific deletion of geranylgeranyl diphosphate synthase 1 (Ggps1) in lung epithelium during fetal lung development resulted in neonatal respiratory distress syndrome-like disease. The knockout mice died at postnatal day 1 of respiratory failure, and the lungs showed compensatory pneumonectasis, pulmonary atelectasis, and hyaline membranes. Subsequently, we proved that lung malformations in Ggps1-deficient mice resulted from the failure of fetal lung branching morphogenesis. Further investigation revealed Ggps1 deletion blocked K-Ras geranylgeranylation and extracellular signal-related kinase 1 or 2/mitogen-activated protein kinase signaling, which in turn disturbed fibroblast growth factor 10 regulation on fetal lung branching morphogenesis. Collectively, our data suggest that GGPPS is essential for maintaining fetal lung branching morphogenesis, which is possibly through regulating K-Ras prenylation. PMID:27106761

  19. Farnesyl Diphosphate Analogues with Aryl Moieties are Efficient Alternate Substrates for Protein Farnesyltransferase

    PubMed Central

    Subramanian, Thangaiah; Pais, June E.; Liu, Suxia; Troutman, Jerry M.; Suzuki, Yuta; Subramanian, Karunai Leela; Fierke, Carol; Andres, Douglas A.; Spielmann, H. Peter

    2012-01-01

    Farnesylation is an important post-translational modification essential for proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of analogue transfer to peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple turnover activity depends on the analogue structure. Analogues where the first isoprene is replaced by a benzyl group and an analogue where each isoprene is replaced by an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single turnover rate constant for alkylation does depend on the Ca1a2X peptide sequence. These results suggest that the isoprenoid transition state conformation is preferred over the inactive E•FPP• Ca1a2X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for design of novel inhibitors. PMID:22989235

  20. Adenosine deaminase inhibition enhances the inotropic response mediated by A1 adenosine receptor in hyperthyroid guinea pig atrium.

    PubMed

    Kemeny-Beke, Adam; Jakab, Anita; Zsuga, Judit; Vecsernyes, Miklos; Karsai, Denes; Pasztor, Fanni; Grenczer, Maria; Szentmiklosi, Andras Jozsef; Berta, Andras; Gesztelyi, Rudolf

    2007-08-01

    The aim of the present study was to test the hypothesis that inhibition of adenosine deaminase (ADA) enhances the efficiency of signal-transduction of myocardial A1 adenosine receptors in hyperthyroidism. The inotropic response to N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist resistant to ADA, was investigated in the absence or presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an ADA and cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2) inhibitor, or of pentostatin (2'-deoxycoformycin; DCF), an exclusive ADA inhibitor, in left atria isolated from eu- or hyperthyroid guinea pigs. Both ADA inhibitors enhanced the effect of CPA only in hyperthyroid atria. EHNA significantly increased the Emax (mean+/-S.E.M.) from 83.8+/-1.2% to 93.4+/-1.2%, while DCF significantly decreased the logEC50 from -7.5+/-0.07 to -7.83+/-0.07 in hyperthyroid samples. Conversely, EHNA also diminished the logEC50 (from -7.5+/-0.07 to -7.65+/-0.07) and DCF also raised the Emax (from 83.8+/-1.2% to 85.7+/-2%) in hyperthyroidism, but these changes were not significant. In conclusion, ADA inhibition moderately but significantly enhanced the efficiency of A(1) adenosine receptor signaling pathway in the hyperthyroid guinea pig atrium. This suggests that elevated intracellular adenosine level caused by ADA inhibition may improve the suppressed responsiveness to A1 adenosine receptor agonists associated with the hyperthyroid state. Alternatively or in addition, the role of decreased concentration of adenosine degradation products cannot be excluded. Furthermore, in the case of EHNA, inhibition of PDE2 also appears to contribute to the enhanced A1 adenosine receptor signaling in the hyperthyroid guinea pig atrium. PMID:17574432

  1. Interstitial adenosine concentration is increased by dipyridamole

    SciTech Connect

    Gorman, M.W.; Wangler, R.D.; DeWitt, D.F.; Wang, C.Y.; Bassingthwaighte, J.B.; Sparks, H.V.

    1986-03-01

    The authors used the multiple indicator dilution technique to observe the capillary transport of adenosine (ADO) in isolated guinea pig hearts. Radiolabelled albumin, sucrose and ADO were injected on the arterial side and measured in venous samples collected during the following 20 seconds. Transport parameters calculated from these data include permeability-surface area products (PS) for transendothelial diffusion, endothelial cell (EC) uptake at the lumenal and ablumenal membranes, and EC metabolism. With simultaneous measurements of arterial and venous ADO concentrations and flow, the authors calculated the steady-state interstitial fluid (ISF) ADO concentration. Under control conditions the venous ADO concentration was 7.1 +/- 2.8 nM. The calculated ISF concentration depends on whether they assume the venous ADO comes from the ISF, or directly from ECs. These ISF concentrations are 25 +/- 12 nM and 9.8 +/- 4.0 nM, respectively. During dipyridamole infusion (10 uM) the EC transport parameters became nearly zero. Venous and ISF ADO concentrations increased to 33 +/- 8.9 nM and 169 +/- 42 nM, respectively. The authors conclude that the ISF ADO concentration is 1.5-4 fold higher than the venous concentration at rest, and the ISF concentration increases greatly with dipyridamole.

  2. Role of A3 adenosine receptor in diabetic neuropathy.

    PubMed

    Yan, Heng; Zhang, Enshui; Feng, Chang; Zhao, Xin

    2016-10-01

    Neuropathy is the most common diabetic complication. Although the A1 and A2A adenosine receptors are important pharmacological targets in alleviating diabetic neuropathy, the role of the A3 adenosine receptor remains unknown. Because the A3 adenosine receptor regulates pain induced by chronic constriction injury or chemotherapy, its stimulation might also attenuate diabetic neuropathy. This study examines the effects of systemic treatment with the A3 adenosine receptor agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA) on diabetic neuropathy and explores the putative mechanisms underlying its pharmacological effects. We show that IB-MECA alleviated mechanical hyperalgesia and thermal hypoalgesia in mice 2 weeks but not 4 weeks after streptozocin (STZ) treatment. Furthermore, IB-MECA prevented the reduction in sciatic motor nerve conduction velocity and sensory nerve conduction velocity in diabetic mice 2 weeks but not 4 weeks after STZ treatment. Similarly, IB-MECA inhibited the activation of nuclear factor-κB and decreased the generation of tumor necrosis factor-α in the spinal cord of mice 2 weeks but not 4 weeks after STZ treatment. These phenomena were associated with reduction of A3 adenosine receptor expression in the spinal cord after long-term diabetes. Our results suggest that the A3 adenosine receptor plays a critical role in regulating diabetic neuropathy and that reduction in A3 adenosine receptor expression/function might contribute to the progression of diabetic neuropathy. © 2016 Wiley Periodicals, Inc. PMID:27319979

  3. Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

    The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism. PMID:21511036

  4. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling

    PubMed Central

    Wen, Jiaming; Jiang, Xianzhen; Dai, Yingbo; Zhang, Yujin; Tang, Yuxin; Sun, Hong; Mi, Tiejuan; Phatarpekar, Prasad V.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2010-01-01

    Priapism is a condition of persistent penile erection in the absence of sexual excitation. Of men with sickle cell disease (SCD), 40% display priapism. The disorder is a dangerous and urgent condition, given its association with penile fibrosis and eventual erectile dysfunction. Current strategies to prevent its progression are poor because of a lack of fundamental understanding of the molecular mechanisms for penile fibrosis in priapism. Here we demonstrate that increased adenosine is a novel causative factor contributing to penile fibrosis in two independent animal models of priapism, adenosine deaminase (ADA)-deficient mice and SCD transgenic mice. An important finding is that chronic reduction of adenosine by ADA enzyme therapy successfully attenuated penile fibrosis in both mouse models, indicating an essential role of increased adenosine in penile fibrosis and a novel therapeutic possibility for this serious complication. Subsequently, we identified that both mice models share a similar fibrotic gene expression profile in penile tissue (including procollagen I, TGF-β1, and plasminogen activator inhibitor-1 mRNA), suggesting that they share similar signaling pathways for progression to penile fibrosis. Thus, in an effort to decipher specific cell types and underlying mechanism responsible for adenosine-mediated penile fibrosis, we purified corpus cavernosal fibroblast cells (CCFCs), the major cell type involved in this process, from wild-type mice. Quantitative RT-PCR showed that the major receptor expressed in these cells is the adenosine receptor A2BR. Based on this fact, we further purified CCFCs from A2BR-deficient mice and demonstrated that A2BR is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-β functions downstream of the A2BR to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A2BR signaling and

  5. Effect of adenosine on the growth of human T-lymphocyte leukemia cell line MOLT-4.

    PubMed

    Streitová, Denisa; Weiterová, Lenka; Hofer, Michal; Holá, Jirina; Horváth, Viktor; Kozubík, Alois; Znojil, Vladimír

    2007-09-01

    Adenosine has been observed to suppress the growth of MOLT-4 human leukemia cells in vitro. Changes in the cell cycle, especially increased percentage of cells in S phase, prolonged generation time, and induction of apoptosis at higher adenosine concentrations have been found to be responsible for the growth suppression. Dipyridamole, a drug inhibiting the cellular uptake of adenosine, reversed partially but significantly the adenosine-induced growth suppression. It follows from these results that the action of adenosine on the MOLT-4 cells comprises its cellular uptake and intracellular operation. These findings present new data on anticancer efficacy of adenosine. PMID:17882653

  6. Substrate-Induced Change in the Quaternary Structure of Type 2 Isopentenyl Diphosphate Isomerase from Sulfolobus shibatae

    PubMed Central

    Nakatani, Hitomi; Goda, Shuichiro; Unno, Hideaki; Nagai, Takuya; Yoshimura, Tohru

    2012-01-01

    Type 2 isopentenyl diphosphate isomerase catalyzes the interconversion between two active units for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylallyl diphosphate, in almost all archaea and in some bacteria, including human pathogens. The enzyme is a good target for discovery of antibiotics because it is essential for the organisms that use only the mevalonate pathway to produce the active isoprene units and because humans possess a nonhomologous isozyme, type 1 isopentenyl diphosphate isomerase. However, type 2 enzymes were reportedly inhibited by mechanism-based drugs for the type 1 enzyme due to their surprisingly similar reaction mechanisms. Thus, a different approach is now required to develop new inhibitors specific to the type 2 enzyme. X-ray crystallography and gel filtration chromatography revealed that the enzyme from a thermoacidophilic archaeon, Sulfolobus shibatae, is in the octameric state at a high concentration. Interestingly, a part of the regions that are involved in the substrate binding in the previously reported tetrameric structures is integral to the formation of the tetramer-tetramer interface in the substrate-free octameric structure. Site-directed mutagenesis at such regions resulted in stabilization of the tetramer. Small-angle X-ray scattering, tryptophan fluorescence, and dynamic light scattering analyses showed that substrate binding causes the dissociation of an octamer into tetramers. This property, i.e., incompatibility between octamer formation and substrate binding, might provide clues to develop new specific inhibitors of the archaeal enzyme. PMID:22505674

  7. Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

    PubMed

    Tepper, A D; Dammann, H; Bominaar, A A; Véron, M

    1994-12-23

    Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase. PMID:7798215

  8. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    PubMed

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia. PMID:23800667

  9. Enzymatic synthesis of acyclic nucleoside thiophosphonate diphosphates: effect of the α-phosphorus configuration on HIV-1 RT activity.

    PubMed

    Priet, Stéphane; Roux, Loic; Saez-Ayala, Magali; Ferron, François; Canard, Bruno; Alvarez, Karine

    2015-05-01

    The acyclic nucleosides thiophosphonates (9-[2-(thiophosphonomethoxy)ethyl]adenine (S-PMEA) and (R)-9-[2-(thiophosphonomethoxy)propyl]adenine (S-PMPA), exhibit antiviral activity against HIV-1, -2 and HBV. Their diphosphate forms S-PMEApp and S-PMPApp, synthesized as stereoisomeric mixture, are potent inhibitors of wild-type (WT) HIV-1 RT. Understanding HIV-1 RT stereoselectivity, however, awaits resolution of the diphosphate forms into defined stereoisomers. To this aim, thiophosphonate monophosphates S-PMEAp and S-PMPAp were synthesized and used in a stereocontrolled enzyme-catalyzed phosphoryl transfer reaction involving either nucleoside diphosphate kinase (NDPK) or creatine kinase (CK) to obtain thiophosphonate diphosphates as separated isomers. We then quantified substrate preference of recombinant WT HIV-1 RT toward pure stereoisomers using in vitro steady-state kinetic analyses. The crystal structure of a complex between Dictyostelium NDPK and S-PMPApp at 2.32Å allowed to determine the absolute configuration at the α-phosphorus atom in relation to the stereo-preference of studied enzymes. The RP isomer of S-PMPApp and S-PMEApp are the preferred substrate over SP for both NDPK and HIV-1 RT. PMID:25766862

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

    PubMed

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

    2015-02-01

    This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the determination of adenosine in urine. PMID:25604821

  11. Unpredictable Chronic Stress Alters Adenosine Metabolism in Zebrafish Brain.

    PubMed

    Zimmermann, F F; Altenhofen, S; Kist, L W; Leite, C E; Bogo, M R; Cognato, G P; Bonan, C D

    2016-05-01

    Stress is considered a risk factor for several human disorders. Despite the broad knowledge of stress responses in mammals, data on the relationship between unpredictable chronic stress (UCS) and its effects on purinergic signaling are limited. ATP hydrolysis by ectonucleotidases is an important source of adenosine, and adenosine deaminase (ADA) contributes to the control of the nucleoside concentrations. Considering that some stress models could affect signaling systems, the objective of this study was to investigate whether UCS alters ectonucleotidase and ADA pathway in zebrafish brain. Additionally, we analyzed ATP metabolism as well as ada1, ada2.1, ada2.2, adaL, and adaasi gene expression in zebrafish brain. Our results have demonstrated that UCS did not alter ectonucleotidase and soluble ADA activities. However, ecto-ADA activity was significantly decreased (26.8%) in brain membranes of animals exposed to UCS when compared to the control group. Quantitative reverse transcription PCR (RT-PCR) analysis did not show significant changes on ADA gene expression after the UCS exposure. The brain ATP metabolism showed a marked increase in adenosine levels (ADO) in animals exposed to UCS. These data suggest an increase on extracellular adenosine levels in zebrafish brain. Since this nucleoside has neuromodulatory and anxiolytic effects, changes in adenosine levels could play a role in counteracting the stress, which could be related to a compensatory mechanism in order to restore the homeostasis. PMID:26081145

  12. Dicinnamoylquinides in roasted coffee inhibit the human adenosine transporter.

    PubMed

    de Paulis, Tomas; Schmidt, Dennis E; Bruchey, Aleksandra K; Kirby, Michael T; McDonald, Michael P; Commers, Patricia; Lovinger, David M; Martin, Peter R

    2002-05-10

    Preliminary screening of a minor, non-xanthine constituent of roasted coffee, 3,4-diferuloyl-1,5-quinolactone (DIFEQ), showed inhibition of the adenosine transporter at low micromolar concentration. DIFEQ is a neutral derivative of the chlorogenic acids, i.e. isomeric mono- and di-substituted coumaroyl-, caffeoyl-, and feruloyl-esters of quinic acid, formed in the roasting process of coffee. Displacement of the adenosine transporter antagonist [(3)H](S)-(nitrobenzyl)-6-thioinosine binding by DIFEQ in cultured U-937 cell preparations, expressing the human adenosine transporter protein (hENT1), showed a K(i) of 0.96+/-0.13 microM. Extracts of regular and decaffeinated coffee showed binding activities equivalent to 30-40 mg DIFEQ per three cups of coffee. Acute administration of a high dose of DIFEQ (100 mg/kg i.p.) reduced open field locomotion in mice for 20 min in correlation with brain levels of DIFEQ. Both 3,4-dicaffeoyl-1,5-quinide and 3,4-dicoumaroyl-1,5-quinide, two close structural analogs of DIFEQ also present in roasted coffee, showed similar affinities for the adenosine transporter, while the corresponding 3- and 4-mono caffeoyl- and feruloyl-quinides were one to two orders of magnitudes less active. This suggests that 3,4-dicinnamoyl-1,5-quinides in coffee could have the potential to raise extra-cellular adenosine levels, thereby counteracting the stimulant effect of caffeine. PMID:12065074

  13. Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

    PubMed

    Liang, Dongchun; Zuo, Aijun; Zhao, Ronglan; Shao, Hui; Kaplan, Henry J; Sun, Deming

    2016-03-15

    Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies. PMID:26856700

  14. Prehospital use of adenosine by ambulance services in the Netherlands

    PubMed Central

    Adams, R.; Bon, V.

    2003-01-01

    Background The prehospital use of adenosine in the treatment of supraventricular arrhythmias has recently been implemented in standard ambulance care. However, establishing the origin and nature of the arrhythmia with certainty is an absolute requirement for using adenosine. Methods The ability of the ambulance nurse to predict supraventricular arrhythmias and the necessity of prehospital treatment of arrhythmias in general was evaluated. To do this, cardiologists at the Academic Medical Centre of Amsterdam were consulted and a literature search by means of an electronic search in Pubmed was performed. The search was complemented by a second survey concerning antagonists of adenosine using the keywords: adenosine and theophylline. Moreover, the Ambulance Nurse textbook, the National Protocol for Ambulance Care as well as the explanatory memorandum to the protocol were consulted. Results No strong indication for the prehospital use of adenosine was found, while detrimental effects of the drug can occur. There is no literature showing the ability of ambulance staff to correctly interpret complex cardiac arrhythmias in the Netherlands; the current ambulance protocol does not prevent an incorrect choice of therapy and medication. Conclusion It is strongly advised against using antiarrhythmic medication for the treatment of tachycardias in a prehospital setting if this treatment can be postponed to the hospital environment. PMID:25696211

  15. Adenosine signaling and the regulation of chronic lung disease

    PubMed Central

    Zhou, Yang; Schneider, Daniel J.; Blackburn, Michael R.

    2009-01-01

    Chronic lung diseases such as asthma, chronic obstructive pulmonary disease and interstitial lung disease are characterized by inflammation and tissue remodeling processes that compromise pulmonary function. Adenosine is produced in the inflamed and damaged lung where it plays numerous roles in the regulation of inflammation and tissue remodeling. Extracellular adenosine serves as an autocrine and paracrine signaling molecule by engaging cell surface adenosine receptors. Preclinical and cellular studies suggest that adenosine plays an anti-inflammatory role in processes associated with acute lung disease, where activation of the A2AR and A2BR have promising implications for the treatment of these disorders. In contrast, there is growing evidence that adenosine signaling through the A1R, A2BR and A3R may serve pro-inflammatory and tissue remodeling functions in chronic lung diseases. This review discusses the current progress of research efforts and clinical trials aimed at understanding the complexities of this signaling pathway as they pertain to the development of treatment strategies for chronic lung diseases. PMID:19426761

  16. Antagonism by theophylline of respiratory inhibition induced by adenosine.

    PubMed

    Eldridge, F L; Millhorn, D E; Kiley, J P

    1985-11-01

    The effects on respiration of an analogue of adenosine, L-2-N6-(phenylisopropyl)adenosine (PIA), and of the methylxanthine, theophylline, were determined in 19 vagotomized glomectomized cats whose end-tidal PCO2 was kept constant by means of a servo-controlled ventilator. Integrated phrenic nerve activity was used to represent respiratory output. Our results show that PIA, whether given systemically or into the third cerebral ventricle, depressed respiration. Systemically administered theophylline stimulated respiration. Theophylline given intravenously, or into the third ventricle not only reversed the depressive effects of previously administered PIA but caused further increases of respiration above the control level. Prior systemic administration of theophylline blocked both respiratory and hypotensive effects of subsequently administered PIA. Effects of either agent on medullary extracellular fluid pH did not explain the results. We conclude that the adenosine analogue PIA, acts to inhibit neurons in the brain that are involved in the control of respiration and that its effects are blocked by theophylline. We suggest that adenosine acts as a tonic modulator of respiration and that theophylline stimulates breathing by competitive antagonism of adenosine at neuronal receptor sites. PMID:4066573

  17. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    PubMed Central

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005 ▶), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P213, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities. PMID:16511098

  18. Synthetic Pathway for Production of Five-Carbon Alcohols from Isopentenyl Diphosphate

    PubMed Central

    Chou, Howard H.

    2012-01-01

    Synthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell. PMID:22941086

  19. Structure Conservation and Differential Expression of Farnesyl Diphosphate Synthase Genes in Euphorbiaceous Plants

    PubMed Central

    Guo, Dong; Li, Hui-Liang; Peng, Shi-Qing

    2015-01-01

    Farnesyl diphosphate synthase (FPS) is a key enzyme of isoprenoids biosynthesis. However, knowledge of the FPSs of euphorbiaceous species is limited. In this study, ten FPSs were identified in four euphorbiaceous plants. These FPSs exhibited similar exon/intron structure. The deduced FPS proteins showed close identities and exhibited the typical structure of plant FPS. The members of the FPS family exhibit tissue expression patterns that vary among several euphorbiaceous plant species under normal growth conditions. The expression profiles reveal spatial and temporal variations in the expression of FPSs of different tissues from Euphorbiaceous plants. Our results revealed wide conservation of FPSs and diverse expression in euphorbiaceous plants during growth and development. PMID:26389894

  20. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    SciTech Connect

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V.

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  1. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    PubMed

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes. PMID:26883346

  2. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    PubMed

    Boissier, Fanny; Georgescauld, Florian; Moynié, Lucile; Dupuy, Jean-William; Sarger, Claude; Podar, Mircea; Lascu, Ioan; Giraud, Marie-France; Dautant, Alain

    2012-06-01

    The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function. PMID:22467275

  3. Repeated febrile convulsions impair hippocampal neurons and cause synaptic damage in immature rats: neuroprotective effect of fructose-1,6-diphosphate

    PubMed Central

    Zhou, Jianping; Wang, Fan; Zhang, Jun; Gao, Hui; Yang, Yufeng; Fu, Rongguo

    2014-01-01

    Fructose-1,6-diphosphate is a metabolic intermediate that promotes cell metabolism. We hypothesize that fructose-1,6-diphosphate can protect against neuronal damage induced by febrile convulsions. Hot-water bathing was used to establish a repetitive febrile convulsion model in rats aged 21 days, equivalent to 3–5 years in humans. Ninety minutes before each seizure induction, rats received an intraperitoneal injection of low- or high-dose fructose-1,6-diphosphate (500 or 1,000 mg/kg, respectively). Low- and high-dose fructose-1,6-diphosphate prolonged the latency and shortened the duration of seizures. Furthermore, high-dose fructose-1,6-diphosphate effectively reduced seizure severity. Transmission electron microscopy revealed that 24 hours after the last seizure, high-dose fructose-1,6-diphosphate reduced mitochondrial swelling, rough endoplasmic reticulum degranulation, Golgi dilation and synaptic cleft size, and increased synaptic active zone length, postsynaptic density thickness, and synaptic interface curvature in the hippocampal CA1 area. The present findings suggest that fructose-1,6-diphosphate is a neuroprotectant against hippocampal neuron and synapse damage induced by repeated febrile convulsion in immature rats. PMID:25206915

  4. Current status of A1 adenosine receptor allosteric enhancers.

    PubMed

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Moorman, Allan R; Borea, Pier Andrea; Varani, Katia

    2015-01-01

    Adenosine is an ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1, A2A, A2B and A3 adenosine receptors (ARs). Allosteric enhancers to A1ARs may represent novel therapeutic agents because they increase the activity of these receptors by mediating a shift to their active form in the A1AR-G protein ternary complex. In this manner, they are able to amplify the action of endogenous adenosine, which is produced in high concentrations under conditions of metabolic stress. A1AR allosteric enhancers could be used as a justifiable alternative to the exogenous agonists that are characterized by receptor desensitization and downregulation. In this review, an analysis of some of the most interesting allosteric modulators of A1ARs has been reported. PMID:26144263

  5. Adenosine deaminase--the non-invasive marker of tuberculosis.

    PubMed

    Pal, Shyamali; Gupta, Sanjoy

    2012-01-01

    Pulmonary tuberculosis is the India's biggest health problem especially in rural areas. A quick and dependable investigation is absolutely essential. Adenosine deaminase was estimated from the biological fluids (ascitic/pleural/CSF) with the help of the kit obtained from Tulip India Pvt Ltd. The method is based on the principle of Galati & Giusti colorimetric method. The method is simple, inexpensive and results are also reproducible. Elevation of adenosine deaminase has shown high specificity in all biological fluids. As the estimation principle is based on synthesis of ammonia so there is limitation of the procedure when the site is kidney. Similarly if the site is skin, as fluid cannot be collected from the site, adenosine deaminase estimation is also not possible. PMID:23029824

  6. Stability of Diluted Adenosine Solutions in Polyolefin Infusion Bags

    PubMed Central

    Almagambetova, Elise; Hutchinson, David; Blais, Danielle M.; Zhao, Fang

    2013-01-01

    Background: Intravenous or intracoronary adenosine is used in the cardiac catherization lab to achieve maximal coronary blood flow and determine fractional flow reserve. Objective: To determine the stability of adenosine 10 and 50 µg/mL in either 0.9% sodium chloride injection or 5% dextrose injection in polyolefin infusion bags stored at 2 temperatures, refrigeration (2°C-8°C) or controlled room temperature (20°C-25°C). Methods: Adenosine 10 µg/mL and 50 µg/mL solutions were prepared in 50 mL polyolefin infusion bags containing 0.9% sodium chloride injection or 5% dextrose injection and stored at controlled room temperature or under refrigeration. Each combination of concentration, diluent, and storage was prepared in triplicate. Samples were assayed using stability-indicating, reversed-phase high-performance liquid chromatography immediately at time 0 and at 24 hours, 48 hours, 7 days, and 14 days. Stability was defined as retaining 90% to 110% of the initial adenosine concentration. The samples were also visually inspected against a light background for clarity, color, and the presence of particulate matter. Results: After 14 days, all samples retained 99% to 101% of the initial adenosine concentration. No considerable change in pH or visual appearance was noted. The stability data indicated no significant loss of drug due to chemical degradation or physical interactions during storage. Conclusion: Adenosine solutions of 10 and 50 µg/mL were stable for at least 14 days in 50 mL polyolefin infusion bags of 0.9% sodium chloride injection or 5% dextrose injection stored at controlled room temperature and refrigerated conditions. PMID:24421510

  7. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  8. Why do premature newborn infants display elevated blood adenosine levels?

    PubMed

    Panfoli, Isabella; Cassanello, Michela; Bruschettini, Matteo; Colella, Marina; Cerone, Roberto; Ravera, Silvia; Calzia, Daniela; Candiano, Giovanni; Ramenghi, Luca

    2016-05-01

    Our preliminary data show high levels of adenosine in the blood of very low birth weight (VLBW) infants, positively correlating to their prematurity (i.e. body weight class). This prompted us to look for a mechanism promoting such impressive adenosine increase. We hypothesized a correlation with oxygen challenge. In fact, it is recognized that either oxygen lack or its excess contribute to the pathogenesis of the injuries of prematurity, such as retinopathy (ROP) and periventricular white matter lesions (PWMI). The optimal concentration of oxygen for resuscitation of VLBW infants is currently under revision. We propose that the elevated adenosine blood concentrations of VLBW infants recognizes two sources. The first could be its activity-dependent release from unmyelinated brain axons. Adenosine in this respect would be an end-product of the hypometabolic VLBW newborn unmyelinated axon intensely firing in response to the environmental stimuli consequent to premature birth. Adenosine would be eventually found in the blood due to blood-brain barrier immaturity. In fact, adenosine is the primary activity-dependent signal promoting differentiation of premyelinating oligodendrocyte progenitor cells (OPC) into myelinating cells in the Central Nervous System, while inhibiting their proliferation and inhibiting synaptic function. The second, would be the ecto-cellular ATP synthesized by the endothelial cell plasmalemma exposed to ambient oxygen concentrations due to premature breathing, especially in lung. ATP would be rapidly transformed into adenosine by the ectonucleotidase activities such as NTPDase I (CD39), and NT5E (CD73). An ectopic extra-mitochondrial aerobic ATP synthetic ability was reported in many cell plasma-membranes, among which endothelial cells. The potential implications of the cited hypotheses for the neonatology area would be great. The amount of oxygen administration for reviving of newborns would find a molecular basis for its assessment. VLBW

  9. Cyclic adenosine monophosphate phosphodiesterase in brain: effect on anxiety.

    PubMed

    Beer, B; Chasin, M; Clody, D E; Vogel, J R

    1972-04-28

    Drugs that reduce anxiety may be mediated by cyclic adenosine monophosphate in the brain because (i) potent anxiety-reducing drugs are also potent inhibitors of brain phosphodiesterase activity; (ii) dibutyryl cyclic adenosine monophosphate has the ability to reduce anxiety; (iii) the methylxanthines show significant anxiety-reducing effects; (iv) theophylline and chlordiazepoxide produce additive anxiety-reducing activity; and (v) there is a significant correlation between the anxiety-reducing property of drugs and their ability to inhibit phosphodiesterase activity in the brain. PMID:4402069

  10. Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

    PubMed Central

    Sayós, J; Solsona, C; Mallol, J; Lluis, C; Franco, R

    1994-01-01

    Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney. PMID:8110185

  11. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning.

    PubMed Central

    Heurteaux, C; Lauritzen, I; Widmann, C; Lazdunski, M

    1995-01-01

    Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels. Images Fig. 2 Fig. 3 PMID:7753861

  12. ATP-Sensitive K+ Channels Regulate the Concentrative Adenosine Transporter CNT2 following Activation by A1 Adenosine Receptors

    PubMed Central

    Duflot, Sylvie; Riera, Bárbara; Fernández-Veledo, Sonia; Casadó, Vicent; Norman, Robert I.; Casado, F. Javier; Lluís, Carme; Franco, Rafael; Pastor-Anglada, Marçal

    2004-01-01

    This study describes a novel mechanism of regulation of the high-affinity Na+-dependent adenosine transporter (CNT2) via the activation of A1 adenosine receptors (A1R). This regulation is mediated by the activation of ATP-sensitive K+ (KATP) channels. The high-affinity Na+-dependent adenosine transporter CNT2 and A1R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (−)-N6-(2-phenylisopropyl)adenosine is fully inhibited by KATP channel blockers and mimicked by a KATP channel opener. A1R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A1R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A1R, CNT2, and KATP channels. These results also suggest that the activation of KATP channels under metabolic stress can be mediated by the activation of A1R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for KATP channels. PMID:15024061

  13. The adenosine metabolite inosine is a functional agonist of the adenosine A2A receptor with a unique signaling bias.

    PubMed

    Welihinda, Ajith A; Kaur, Manmeet; Greene, Kelly; Zhai, Yongjiao; Amento, Edward P

    2016-06-01

    Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A2A receptor (A2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A2AR and subsequent activation of downstream signaling events. Inosine-mediated A2AR activation leads to cAMP production with an EC50 of 300.7μM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38μM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A2AR activation in vivo. PMID:26903141

  14. Adenosine-dependent activation of tyrosine hydroxylase is defective in adenosine kinase-deficient PC12 cells.

    PubMed Central

    Erny, R; Wagner, J A

    1984-01-01

    (R)-N6-Phenylisopropyladenosine (PIA) stimulates dopa production 3- to 5-fold in PC12 cells, with a half-maximal effective concentration (EC50) of 50 nM. This increase can be explained by a stable activation of tyrosine hydroxylase [TyrOHase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] when it is phosphorylated by a cAMP-dependent protein kinase. The activation of TyrOHase is mediated by the adenosine-dependent activation of adenylate cyclase (EC50 = 600 nM). PIA (10 microM) is as effective as cholera toxin or dibutyryl cAMP in activating TyrOHase in wild-type cells. Adenosine kinase-deficient mutants of PC12 were found to be resistant to PIA-dependent activation of TyrOHase (EC50 = 100-1000 nM). This phenomenon was explored in detail in one adenosine kinase-deficient mutant and was shown to occur because the mutant was resistant to the adenosine-dependent activation of adenylate cyclase. In this mutant, TyrOHase was activated 14-fold by cholera toxin, suggesting that activated TyrOHase is about 14 times as active as unactivated TyrOHase. These studies with kinase-deficient PC12 cells provide genetic evidence that adenosine-dependent activation of TyrOHase is mediated by acute increases in cAMP. When the adenosine receptor found on PC12 cells is expressed in vivo, it might function as either a presynaptic (i.e., localized on the nerve terminal) or a postsynaptic (i.e., localized on the cell body or dendrite) receptor that regulates rates of transmitter synthesis in response to cell activity. PMID:6146982

  15. Anticancer effect of adenosine on gastric cancer via diverse signaling pathways

    PubMed Central

    Tsuchiya, Ayako; Nishizaki, Tomoyuki

    2015-01-01

    Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer. PMID:26494951

  16. Anticancer effect of adenosine on gastric cancer via diverse signaling pathways.

    PubMed

    Tsuchiya, Ayako; Nishizaki, Tomoyuki

    2015-10-21

    Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer. PMID:26494951

  17. Neuronal transporter and astrocytic ATP exocytosis underlie activity-dependent adenosine release in the hippocampus

    PubMed Central

    Wall, Mark J; Dale, Nicholas

    2013-01-01

    The neuromodulator adenosine plays an important role in many physiological and pathological processes within the mammalian CNS. However, the precise mechanisms of how the concentration of extracellular adenosine increases following neural activity remain contentious. Here we have used microelectrode biosensors to directly measure adenosine release induced by focal stimulation in stratum radiatum of area CA1 in mouse hippocampal slices. Adenosine release was both action potential and Ca2+ dependent and could be evoked with low stimulation frequencies and small numbers of stimuli. Adenosine release required the activation of ionotropic glutamate receptors and could be evoked by local application of glutamate receptor agonists. Approximately 40% of stimulated-adenosine release occurred by translocation of adenosine via equilibrative nucleoside transporters (ENTs). This component of release persisted in the presence of the gliotoxin fluoroacetate and thus results from the direct release of adenosine from neurons. A reduction of adenosine release in the presence of NTPDase blockers, in slices from CD73−/− and dn-SNARE mice, provides evidence that a component of adenosine release arises from the extracellular metabolism of ATP released from astrocytes. This component of release appeared to have slower kinetics than the direct ENT-mediated release of adenosine. These data suggest that activity-dependent adenosine release is surprisingly complex and, in the hippocampus, arises from at least two distinct mechanisms with different cellular sources. PMID:23713028

  18. Adenosine transporters and receptors: key elements for retinal function and neuroprotection.

    PubMed

    Dos Santos-Rodrigues, Alexandre; Pereira, Mariana R; Brito, Rafael; de Oliveira, Nádia A; Paes-de-Carvalho, Roberto

    2015-01-01

    Adenosine is an important neuroactive substance in the central nervous system, including in the retina where subclasses of adenosine receptors and transporters are expressed since early stages of development. Here, we review some evidence showing that adenosine plays important functions in the mature as well as in the developing tissue. Adenosine transporters are divided into equilibrative and concentrative, and the major transporter subtype present in the retina is the ENT1. This transporter is responsible for a bidirectional transport of adenosine and the uptake or release of this nucleoside appears to be regulated by different signaling pathways that are also controlled by activation of adenosine receptors. Adenosine receptors are also key players in retina physiology regulating a variety of functions in the mature and developing tissue. Regulation of excitatory neurotransmitter release and neuroprotection are the main functions played be adenosine in the mature tissue, while regulation of cell survival and neurogenesis are some of the functions played by adenosine in developing retina. Since adenosine is neuroprotective against excitotoxic and metabolic dysfunctions observed in neurological and ocular diseases, the search for adenosine-related drugs regulating adenosine transporters and receptors can be important for advancement of therapeutic strategies against these diseases. PMID:25817878

  19. Amelioration of adriamycin and daunorubicin myocardial toxicity by adenosine.

    PubMed

    Newman, R A; Hacker, M P; Krakoff, I H

    1981-09-01

    Primary cultures of rat myocardial cells were used to investigate the dose and time-dependent cellular enzyme release induced by either Adriamycin or daunorubicin, Concentrations of either anthracycline (1.8 or 18 microM) produced significant release of creatine phosphokinase and lactic dehydrogenase from myocardial cells within 24 hr of exposure without a detectable decrease in cell viability. Preincubation of the myocardial cells with varying concentrations of adenosine (10 microM to 1 mM) for 24 hr prior to the addition of anthracycline decreased or prevented drug-induced enzyme release. Other putative myocardial protectants, i.e., N-acetyl-L-cysteine, alpha-tocopherol, or carnitine, were ineffective in preventing anthracycline-induced enzyme release. Although adenosine was an effective myocardial protectant, it had no significant effect on cellular uptake of daunorubicin, nor did adenosine adversely affect the oncolytic activity of daunorubicin against L1210 leukemia cells in vitro. Anthramycin, another oncolytic agent having reported cardiotoxic effects, was also tested in the in vitro system. With this drug, however, no enzyme release was detected at less than lethal doses nor did adenosine have any protective potential against the toxicity of anthramycin. Finally, Adriamycin caused no significant lactic dehydrogenase release when incubated at 1.8 or 18 microM with H9c2 cells, a cell line having primarily skeletal muscle characteristics. This result suggests a specific toxicity of anthracyclines for myocardial but not skeletal muscle cells. PMID:7260911

  20. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  1. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  2. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  3. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  4. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  5. CD39/Adenosine Pathway Is Involved in AIDS Progression

    PubMed Central

    Limou, Sophie; Younas, Mehwish; Kök, Ayrin; Huë, Sophie; Seddiki, Nabila; Hulin, Anne; Delaneau, Olivier; Schuitemaker, Hanneke; Herbeck, Joshua T.; Mullins, James I.; Muhtarova, Maria; Bensussan, Armand; Zagury, Jean-François; Lelievre, Jean-Daniel; Lévy, Yves

    2011-01-01

    HIV-1 infection is characterized by a chronic activation of the immune system and suppressed function of T lymphocytes. Regulatory CD4+ CD25high FoxP3+CD127low T cells (Treg) play a key role in both conditions. Here, we show that HIV-1 positive patients have a significant increase of Treg-associated expression of CD39/ENTPD1, an ectoenzyme which in concert with CD73 generates adenosine. We show in vitro that the CD39/adenosine axis is involved in Treg suppression in HIV infection. Treg inhibitory effects are relieved by CD39 down modulation and are reproduced by an adenosine-agonist in accordance with a higher expression of the adenosine A2A receptor on patients' T cells. Notably, the expansion of the Treg CD39+ correlates with the level of immune activation and lower CD4+ counts in HIV-1 infected patients. Finally, in a genetic association study performed in three different cohorts, we identified a CD39 gene polymorphism that was associated with down-modulated CD39 expression and a slower progression to AIDS. PMID:21750674

  6. Biosynthesis of the Diterpenoid Lycosantalonol via Nerylneryl Diphosphate in Solanum lycopersicum

    PubMed Central

    Matsuba, Yuki; Zi, Jiachen; Jones, A. Daniel; Peters, Reuben J.; Pichersky, Eran

    2015-01-01

    We recently reported that three genes involved in the biosynthesis of monoterpenes in trichomes, a cis-prenyltransferase named neryl diphosphate synthase 1 (NDPS1) and two terpene synthases (TPS19 and TPS20), are present in close proximity to each other at the tip of chromosome 8 in the genome of the cultivated tomato (Solanum lycopersicum). This terpene gene “cluster” also contains a second cis-prenyltransferase gene (CPT2), three other TPS genes, including TPS21, and the cytochrome P450-oxidoreductase gene CYP71BN1. CPT2 encodes a neryneryl diphosphate synthase. Co-expression in E. coli of CPT2 and TPS21 led to the formation of the diterpene lycosantalene, and co-expression in E. coli of CPT2, TPS21 and CYP71BN1 led to the formation of lycosantalonol, an oxidation product of lycosantalene. Here we show that maximal expression of all three genes occurs in the petiolule part of the leaf, but little expression of these genes occurs in the trichomes present on the petiolules. While lycosantalene or lycosantalonol cannot be detected in the petiolules of wild-type plants (or anywhere else in the plant), lycosantalene and lycosantalonol are detected in petiolules of transgenic tomato plants expressing CPT2 under the control of the 35S CaMV promoter. These results suggest that lycosantalene and lycosantalonol are produced in the petiolules and perhaps in other tissues of wild-type plants, but that low rate of synthesis, controlled by the rate-limiting enzyme CPT2, results in product levels that are too low for detection under our current methodology. It is also possible that these compounds are further modified in the plant. The involvement of CPT2, TPS21 and CYP71BN1 in a diterpenoid biosynthetic pathway outside the trichomes, together with the involvement of other genes in the cluster in the synthesis of monoterpenes in trichomes, indicates that this cluster is further evolving into “sub-clusters” with unique biochemical, and likely physiological, roles. PMID

  7. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  8. Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation

    SciTech Connect

    Zhang, Y.; Cao, R; Yin, F; Hudock, M; Guo, R; Song, Y; No, J; Bergan, K; Leon, A; et al,

    2009-01-01

    Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

  9. The adenosine system modulates Toll-like receptor function: basic mechanisms, clinical correlates and translational opportunities

    PubMed Central

    Coombs, Melanie R. Power; Belderbos, Mirjam E.; Gallington, Leighanne C.; Bont, Louis; Levy, Ofer

    2014-01-01

    Adenosine is an endogenous purine metabolite whose concentration in human blood plasma rises from nanomolar to micromolar during stress or hypoxia. Leukocytes express seven-transmembrane adenosine receptors whose engagement modulates Toll-like receptor-mediated cytokine responses, in part via modulation of intracellular cyclic adenosine monophosphate (cAMP). Adenosine congeners are used clinically to treat arrhythmias and apnea of prematurity. Herein we consider the potential of adenosine congeners as innate immune response modifiers to prevent and/or treat infection. PMID:21342073

  10. Adenosine induces G2/M cell-cycle arrest by inhibiting cell mitosis progression.

    PubMed

    Jia, Kun-Zhi; Tang, Bo; Yu, Lu; Cheng, Wei; Zhang, Rong; Zhang, Jian-Fa; Hua, Zi-Chun

    2010-01-01

    Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G-2/M cell-cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis. PMID:19947935

  11. Manipulation of adenosine kinase affects sleep regulation in mice

    PubMed Central

    Palchykova, Svitlana; Winsky-Sommerer, Raphaelle; Shen, Hai-Ying; Boison, Detlev; Gerling, Andrea; Tobler, Irene

    2010-01-01

    Sleep and sleep intensity are enhanced by adenosine and its receptor agonists, while adenosine receptor antagonists induce wakefulness. Adenosine kinase (ADK) is the primary enzyme metabolizing adenosine in adult brain. To investigate whether adenosine metabolism or clearance affects sleep we recorded sleep in mice with engineered mutations in Adk. Adk-tg mice over-express a transgene encoding the cytoplasmic isoform of ADK in the brain, but lack the nuclear isoform of the enzyme. Wild-type mice and Adk+/− mice that have a 50% reduction of the cytoplasmic and the nuclear isoforms of ADK served as controls. Adk-tg mice showed a remarkable reduction of EEG power in low frequencies in all vigilance states and in theta activity (6.25–11 Hz) in REM sleep and waking. Adk-tg mice were awake 58 min more per day than wild-type mice and spent significantly less time in REM sleep (102±3 vs 128±3 min in wild-type). After sleep deprivation slow-wave activity (0.75–4 Hz), the intensity component of NREM sleep, increased significantly less in Adk-tg mice and their slow-wave energy was reduced. In contrast, the vigilance states and EEG spectra of Adk+/− and wild-type mice did not differ. Our data suggest that over-expression of the cytoplasmic isoform of ADK is sufficient to alter sleep physiology. ADK might orchestrate neurotransmitter pathways involved in the generation of EEG oscillations and regulation of sleep. PMID:20881134

  12. Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

    PubMed Central

    Magalhães-Cardoso, Maria Teresa; Ferreirinha, Fátima; Dias, Ana Sofia; Pelletier, Julie

    2014-01-01

    Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders. PMID:25210228

  13. Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis.

    PubMed

    Rai, Avanish; Smita, Shachi S; Singh, Anup Kumar; Shanker, Karuna; Nagegowda, Dinesh A

    2013-09-01

    Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis. PMID:23543438

  14. Modulation of bladder function by luminal adenosine turnover and A1 receptor activation

    PubMed Central

    Prakasam, H. Sandeep; Herrington, Heather; Roppolo, James R.; Jackson, Edwin K.

    2012-01-01

    The bladder uroepithelium transmits information to the underlying nervous and musculature systems, is under constant cyclical strain, expresses all four adenosine receptors (A1, A2A, A2B, and A3), and is a site of adenosine production. Although adenosine has a well-described protective effect in several organs, there is a lack of information about adenosine turnover in the uroepithelium or whether altering luminal adenosine concentrations impacts bladder function or overactivity. We observed that the concentration of extracellular adenosine at the mucosal surface of the uroepithelium was regulated by ecto-adenosine deaminase and by equilibrative nucleoside transporters, whereas adenosine kinase and equilibrative nucleoside transporters modulated serosal levels. We further observed that enriching endogenous adenosine by blocking its routes of metabolism or direct activation of mucosal A1 receptors with 2-chloro-N6-cyclopentyladenosine (CCPA), a selective agonist, stimulated bladder activity by lowering the threshold pressure for voiding. Finally, CCPA did not quell bladder hyperactivity in animals with acute cyclophosphamide-induced cystitis but instead exacerbated their irritated bladder phenotype. In conclusion, we find that adenosine levels at both surfaces of the uroepithelium are modulated by turnover, that blocking these pathways or stimulating A1 receptors directly at the luminal surface promotes bladder contractions, and that adenosine further stimulates voiding in animals with cyclophosphamide-induced cystitis. PMID:22552934

  15. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

  16. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

    PubMed

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. PMID:26971881

  17. Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway

    PubMed Central

    Su, Ping; Tong, Yuru; Cheng, Qiqing; Hu, Yating; Zhang, Meng; Yang, Jian; Teng, Zhongqiu; Gao, Wei; Huang, Luqi

    2016-01-01

    Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes. PMID:26971881

  18. The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes.

    PubMed

    Nemeria, Natalia; Chakraborty, Sumit; Baykal, Ahmet; Korotchkina, Lioubov G; Patel, Mulchand S; Jordan, Frank

    2007-01-01

    Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4'-aminopyrimidine aromatic rings, but only recently has participation of the 4'-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4'-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1',4'-iminopyrimidine and the 4'-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1',4'-iminopyrimidine form. The identification of the 1',4'-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis. PMID:17182735

  19. Neuronal adenosine release, and not astrocytic ATP release, mediates feedback inhibition of excitatory activity

    PubMed Central

    Lovatt, Ditte; Xu, Qiwu; Liu, Wei; Takano, Takahiro; Smith, Nathan A.; Schnermann, Jurgen; Tieu, Kim; Nedergaard, Maiken

    2012-01-01

    Adenosine is a potent anticonvulsant acting on excitatory synapses through A1 receptors. Cellular release of ATP, and its subsequent extracellular enzymatic degradation to adenosine, could provide a powerful mechanism for astrocytes to control the activity of neural networks during high-intensity activity. Despite adenosine's importance, the cellular source of adenosine remains unclear. We report here that multiple enzymes degrade extracellular ATP in brain tissue, whereas only Nt5e degrades AMP to adenosine. However, endogenous A1 receptor activation during cortical seizures in vivo or heterosynaptic depression in situ is independent of Nt5e activity, and activation of astrocytic ATP release via Ca2+ photolysis does not trigger synaptic depression. In contrast, selective activation of postsynaptic CA1 neurons leads to release of adenosine and synaptic depression. This study shows that adenosine-mediated synaptic depression is not a consequence of astrocytic ATP release, but is instead an autonomic feedback mechanism that suppresses excitatory transmission during prolonged activity. PMID:22421436

  20. Hydrophobic statins induce autophagy and cell death in human rhabdomyosarcoma cells by depleting geranylgeranyl diphosphate.

    PubMed

    Araki, Makoto; Maeda, Masatomo; Motojima, Kiyoto

    2012-01-15

    Statins are the most common type of medicine used to treat hypercholesterolemia; however, they are associated with a low incidence of myotoxicity such as myopathy and rhabdomyolysis. The mechanisms for the adverse effects remain to be fully elucidated for safer chronic use and drug development. The results of our earlier work suggested that hydrophobic statins induce autophagy in cultured human rhabdomyosarcoma A204 cells. In this study, we first confirmed the statin-induced autophagy by assessing other criteria, including induced expression of the autophagy-related genes, enhanced protein degradation of autophagy marker protein p62 and electron microscopic observation of induced formation of autophagosome. We next demonstrated that the extent of inhibition of HMG-CoA reductase in the cell is parallel with the ability of a statin to induce autophagy. Thus, the primary activity of statins causes autophagy in A204 cells. Considering the mechanism for the induction, we showed that statins induce autophagy by depleting cellular levels of geranylgeranyl diphosphate (GGPP) mostly through an unknown pathway that does not involve two major small G proteins, Rheb and Ras. Finally, we demonstrated that the ability of statins to induce autophagy parallels their toxicity to A204 cells and that both can be suppressed by GGPP. PMID:22094060

  1. Functional and conformational transitions of mevalonate diphosphate decarboxylase from Bacopa monniera.

    PubMed

    Abbassi, Shakeel; Patel, Krunal; Khan, Bashir; Bhosale, Siddharth; Gaikwad, Sushama

    2016-02-01

    Functional and conformational transitions of mevalonate diphosphate decarboxylase (MDD), a key enzyme of mevalonate pathway in isoprenoid biosynthesis, from Bacopa monniera (BmMDD), cloned and overexpressed in Escherichia coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and Circular dichroism spectroscopy. Native BmMDD is a helix dominant structure with 45% helix and 11% sheets and possesses seven tryptophan residues with two residues exposed on surface, three residues partially exposed and two situated in the interior of the protein. Thermal denaturation of BmMDD causes rapid structural transitions at and above 40°C and transient exposure of hydrophobic residues at 50°C, leading to aggregation of the protein. An acid induced molten globule like structure was observed at pH 4, exhibiting altered but compact secondary structure, distorted tertiary structure and exposed hydrophobic residues. The molten globule displayed different response at higher temperature and similar response to chemical denaturation as compared to the native protein. The surface tryptophans have predominantly positively charged amino acids around them, as indicated by higher KSV for KI as compared to that for CsCl. The native enzyme displayed two different lifetimes, τ1 (1.203±0.036 ns) and τ2 (3.473±0.12 ns) indicating two populations of tryptophan. PMID:26657583

  2. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis.

    PubMed

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  3. Intersubunit Ionic Interactions Stabilize the Nucleoside Diphosphate Kinase of Mycobacterium tuberculosis

    PubMed Central

    Georgescauld, Florian; Moynié, Lucile; Habersetzer, Johann; Cervoni, Laura; Mocan, Iulia; Borza, Tudor; Harris, Pernile; Dautant, Alain; Lascu, Ioan

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg80-Asp93 as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved. PMID:23526954

  4. The effect of cytidine-diphosphate choline (CDP-choline) on brain lipid changes during aging

    SciTech Connect

    De Medio, G.E.; Trovarelli, G.; Piccinin, G.L.; Porcellati, G.

    1984-01-01

    Lipid synthesis has been tested in vivo in different brain areas of 12-month-old male rats. Cortex, striatum, brainstem, and subcortex of brain have been examined. The cerebellum was discarded. Mixtures of (2-/sup 3/H)glycerol and (Me-/sup 14/C)choline were injected into the lateral ventricle of the brain as lipid precursors, and their incorporation into total lipid, water-soluble intermediates and choline-containing phospholipids was examined 1 hr after isotope injection. In another series of experiments cytidine-5'-diphosphate choline (CDP-choline) was injected intraventricularly to the aged rats 10 min before sacrifice with a simultaneous injection, and radioactivity assays were performed as above. Distribution of radioactivity content of CDP-choline among brain areas 10 min after its administration showed a noticeable enrichment of the nucleotide and water-soluble-related compounds in the examined areas, but to a lesser degree in the cerebral cortex. The incorporation of labelled glycerol, which is severely depressed in aged rats in all four areas (Gaiti et al, 1982, 1983), was increased only in the cortex, and apparently decreased in the other areas. This last result is probably due to a dilution effect brought about by the administered cold CDP-choline upon the (/sup 14/C)-containing water-soluble metabolites. As a consequence, the (/sup 3/H)/(/sup 14/C) ratio in total lipid and in isolated phosphatidylcholine and choline plasmalogen increased after CDP-choline treatment.

  5. Cloning and sequence analysis of the Blumea balsamifera DC farnesyl diphosphate synthase gene.

    PubMed

    Pang, Y X; Guan, L L; Wu, L F; Chen, Z X; Wang, K; Xie, X L; Yu, F L; Chen, X L; Zhang, Y B; Jiang, Q

    2014-01-01

    Blumea balsamifera DC is a member of the Compositae family and is frequently used as traditional Chinese medicine. Blumea balsamifera is rich in monoterpenes, which possess a variety of pharmacological activities, such as antioxidant, anti-bacteria, and anti-viral activities. Farnesyl diphosphate synthase (FPS) is a key enzyme in the biosynthetic pathway of terpenes, playing an important regulatory role in plant growth, such as resistance and secondary metabolism. Based on the conserved oligo amino acid residues of published FPS genes from other higher plant species, a cDNA sequence, designated BbFPS, was isolated from B. balsamifera DC using polymerase chain reaction. The clones were an average of 1.6 kb and contained an open reading frame that predicted a polypeptide of 342 amino acids with 89.07% identity to FPS from other plants. The deduced amino acid sequence was dominated by hydrophobic regions and contained 2 highly conserved DDxxD motifs that are essential for proper functioning of FPS. Phylogenetic analysis indicated that FPS grouped with other composite families. Prediction of secondary structure and subcellular localization suggested that alpha helices made up 70% of the amino acids of the sequence. PMID:25501197

  6. The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis.

    PubMed

    Mailloux, Ryan J; Darwich, Rami; Lemire, Joseph; Appanna, Vasu

    2008-04-01

    Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of gamma-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival. PMID:18324728

  7. Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution.

    PubMed

    Ishibashi, Matsujiro; Hayashi, Tomoe; Yoshida, Chiho; Tokunaga, Masao

    2013-07-01

    Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit-subunit interactions through mutations. PMID:23609188

  8. Decaprenyl diphosphate synthase subunit 2 as a prognosis factor in hepatocellular carcinoma

    PubMed Central

    Huang, Wei; Gao, Fei; Li, Kang; Wang, Wen; Lai, Ya-Rou; Tang, Shao-Hui; Yang, Dong-Hua

    2015-01-01

    AIM: To investigate the involvement of decaprenyl diphosphate synthase subunit 2 (PDSS2) in development and progression of human hepatocellular carcinoma (HCC). METHODS: PDSS2 protein expression was examined in well- and poorly differentiated HCC tumor samples. The levels of PDSS2 expression were compared with clinical features and prognosis of HCC patients. The effects of PDSS2 on cell proliferation, cell cycle, apoptosis, cell migration, and invasion in HCC HepG2 cells were also investigated. RESULTS: PDSS2 was downregulated in poorly differentiated cancer samples compared with well-differentiated tumor samples, and the expression level was markedly lower in HCC tissues than in histologically normal tissue adjacent to the cancer. Reduced protein expression was negatively associated with the status of HCC progression. In addition, overexpression of PDSS2 dramatically suppressed cell proliferation and colony formation, and induced apoptosis in HepG2 cells by inducing G1-phase cell-cycle arrest. The migration and invasion capabilities of HepG2 cells were significantly decreased following PDSS2 overexpression. CONCLUSION: Decreased PDSS2 expression is an unfavorable prognostic factor for HCC, and PDSS2 has potent anticancer activity in HCC tissues and HepG2 cells. PMID:25780306

  9. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 degrees C

    SciTech Connect

    Armstrong, Christopher R.; Felmy, Andrew R.; Clark, Sue B.

    2010-11-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO2)(3)(PO4)(2)center dot 4H(2)O) and Na autunite (Na[UO2PO4]center dot xH(2)O) at 23 and 50 degrees C in NaClO4-HClO4 solutions at pC(H+) = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 in solutions were equilibrated at 23 and 50 degrees C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K-sp, for TDT was determined to be -49.7 and -51.3 at 23 and 50 degrees C respectively. log K for Na autunite was determined to be -24.4 (23 degrees C) and -24.1 +/- 0.2 (50 degrees C).

  10. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

    PubMed Central

    Farha, Maya A.; Czarny, Tomasz L.; Myers, Cullen L.; Worrall, Liam J.; French, Shawn; Conrady, Deborah G.; Wang, Yang; Oldfield, Eric; Strynadka, Natalie C. J.; Brown, Eric D.

    2015-01-01

    Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery. PMID:26283394

  11. Evaluation of serum nucleoside diphosphate kinase A for the detection of colorectal cancer

    PubMed Central

    Otero-Estévez, Olalla; De Chiara, Loretta; Barcia-Castro, Leticia; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco Javier; Cubiella, Joaquín; Hernández, Vicent; Martínez-Zorzano, Vicenta Soledad

    2016-01-01

    We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk. PMID:27222072

  12. Identification and Characterization of a Novel Monoterpene Synthase from Soybean Restricted to Neryl Diphosphate Precursor

    PubMed Central

    Li, Kai; Yu, Deyue

    2013-01-01

    Terpenes are important defensive compounds against herbivores and pathogens. Here, we report the identification of a new monoterpene synthase gene, GmNES, from soybean. The transcription of GmNES was up-regulated in soybean plants that were infested with cotton leafworm (Prodenia litura), mechanically wounded or treated with salicylic acid (SA). Gas chromatography-mass spectrometry (GC-MS) analysis revealed that recombinant GmNES enzyme exclusively produced nerol, generated from a newly identified substrate for monoterpene synthase: neryl diphosphate (NPP). This finding indicates that GmNES is a nerol synthase gene in soybean. Subcellular localization using GFP fusions showed that GmNES localized to the chloroplasts. Transgenic tobacco overexpressing GmNES was generated. In dual-choice assays, the GmNES-expressing tobacco lines significantly repelled cotton leafworm. In feeding tests with transgenic plants, the growth and development of cotton leafworm were significantly retarded. This study confirms the ecological role of terpenoids and provides new insights into their metabolic engineering in transgenic plants. PMID:24124526

  13. The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.

    PubMed

    Wille, Georg; Meyer, Danilo; Steinmetz, Andrea; Hinze, Erik; Golbik, Ralph; Tittmann, Kai

    2006-06-01

    Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase. PMID:16680160

  14. Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

    PubMed

    Pateraki, Irene; Kanellis, Angelos K

    2008-05-01

    Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus. PMID:18402992

  15. Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

    PubMed Central

    Lu, Qing; Harrington, Elizabeth O.; Newton, Julie; Casserly, Brian; Radin, Gregory; Warburton, Rod; Zhou, Yang; Blackburn, Michael R.

    2010-01-01

    We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A1 or A3 antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A2A and A2B, respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A2A and A2B receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A2A/A2B receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A2A and A2B agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A2A and A2B receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by α-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the

  16. Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury

    PubMed Central

    Sakhatskyy, Pavlo; Newton, Julie; Shamirian, Paul; Hsiao, Vivian; Curren, Sean; Gabino Miranda, Gustavo Andres; Pedroza, Mesias; Blackburn, Michael R.; Rounds, Sharon

    2013-01-01

    Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases. PMID:23316066

  17. Adenosine-mediated modulation of ventral horn interneurons and spinal motoneurons in neonatal mice.

    PubMed

    Witts, Emily C; Nascimento, Filipe; Miles, Gareth B

    2015-10-01

    Neuromodulation allows neural networks to adapt to varying environmental and biomechanical demands. Purinergic signaling is known to be an important modulatory system in many parts of the CNS, including motor control circuitry. We have recently shown that adenosine modulates the output of mammalian spinal locomotor control circuitry (Witts EC, Panetta KM, Miles GB. J Neurophysiol 107: 1925-1934, 2012). Here we investigated the cellular mechanisms underlying this adenosine-mediated modulation. Whole cell patch-clamp recordings were performed on ventral horn interneurons and motoneurons within in vitro mouse spinal cord slice preparations. We found that adenosine hyperpolarized interneurons and reduced the frequency and amplitude of synaptic inputs to interneurons. Both effects were blocked by the A1-type adenosine receptor antagonist DPCPX. Analysis of miniature postsynaptic currents recorded from interneurons revealed that adenosine reduced their frequency but not amplitude, suggesting that adenosine acts on presynaptic receptors to modulate synaptic transmission. In contrast to interneurons, recordings from motoneurons revealed an adenosine-mediated depolarization. The frequency and amplitude of synaptic inputs to motoneurons were again reduced by adenosine, but we saw no effect on miniature postsynaptic currents. Again these effects on motoneurons were blocked by DPCPX. Taken together, these results demonstrate differential effects of adenosine, acting via A1 receptors, in the mouse spinal cord. Adenosine has a general inhibitory action on ventral horn interneurons while potentially maintaining motoneuron excitability. This may allow for adaptation of the locomotor pattern generated by interneuronal networks while helping to ensure the maintenance of overall motor output. PMID:26311185

  18. Abiotic regioselective phosphorylation of adenosine with borate in formamide.

    PubMed

    Furukawa, Yoshihiro; Kim, Hyo-Joong; Hutter, Daniel; Benner, Steven A

    2015-04-01

    Nearly 40 years ago, Schoffstall and his coworkers used formamide as a solvent to permit the phosphorylation of nucleosides by inorganic phosphate to give nucleoside phosphates, which (due to their thermodynamic instability with respect to hydrolysis) cannot be easily created in water by an analogous phosphorylation (the "water problem" in prebiotic chemistry). More recently, we showed that borate could stabilize certain carbohydrates against degradation (the "asphalt problem"). Here, we combine the two concepts to show that borate can work in formamide to guide the reactivity of nucleosides under conditions where they are phosphorylated. Specifically, reaction of adenosine in formamide with inorganic phosphate and pyrophosphate in the presence of borate gives adenosine-5'-phosphate as the only detectable phosphorylated product, with formylation (as opposed to hydrolysis) being the competing reaction. PMID:25826074

  19. Adenosine: an endogenous mediator in the pathogenesis of psoriasis*

    PubMed Central

    Festugato, Moira

    2015-01-01

    It is known that inflammatory and immune responses protect us from the invasion of micro-organisms and eliminate "wastes" from the injured sites, but they may also be responsible for significant tissue damage. Adenosine, as a purine nucleoside, which is produced in inflamed or injured sites, fulfills its role in limiting tissue damage. Although, it may have a pleiotropic effect, which signals it with a proinflammatory state in certain situations, it can be considered a potent anti-inflammatory mediator. The effects of adenosine, which acts through its receptors on T cell, on mast cell and macrophages, on endothelial cells, on neutrophils and dendritic cells, as they indicate TNF-alpha and cytokines, show that this mediator has a central role in the pathogenesis of psoriasis. The way it acts in psoriasis will be reviewed in this study. PMID:26734868

  20. Pharmacology of the Adenosine A3 Receptor in the Vasculature and Essential Hypertension

    PubMed Central

    Ho, Ming-Fen; Low, Leanne M.; Rose’Meyer, Roselyn B.

    2016-01-01

    Background Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A3 receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A3 receptor agonists in coronary blood vessels using the isolated perfused heart preparation. Methods mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A3 receptor mediated coronary vasodilation in the rat heart. Results Adenosine A3 receptor agonists induced coronary vasodilation. The expression of adenosine A3 receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A3 receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A3 receptor agonists. Conclusions This study demonstrated alterations in the expression of adenosine A3 receptors occurred in a tissue specific mode, and reduced adenosine A3 receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A3 receptor in hypertensive hearts suggest that adenosine A3 receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A3 receptor agonists. PMID:26907173

  1. Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis.

    PubMed

    Alsharif, Khalaf F; Thomas, Mark R; Judge, Heather M; Khan, Haroon; Prince, Lynne R; Sabroe, Ian; Ridger, Victoria C; Storey, Robert F

    2015-08-01

    In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10(-8)M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7%±4.4 vs. control 22.6%±2.4; p<0.01) by acting on the high-affinity A1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10(-8)M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6±6.6 vs. 28.0±6.6; p=0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection. PMID:25869515

  2. Extracellular adenosine levels are associated with the progression and exacerbation of pulmonary fibrosis.

    PubMed

    Luo, Fayong; Le, Ngoc-Bao; Mills, Tingting; Chen, Ning-Yuan; Karmouty-Quintana, Harry; Molina, Jose G; Davies, Jonathan; Philip, Kemly; Volcik, Kelly A; Liu, Hong; Xia, Yang; Eltzschig, Holger K; Blackburn, Michael R

    2016-02-01

    Idiopathic pulmonary fibrosis is a devastating lung disease with limited treatment options. The signaling molecule adenosine is produced in response to injury and serves a protective role in early stages of injury and is detrimental during chronic stages of disease such as seen in lung conditions such as pulmonary fibrosis. Understanding the association of extracellular adenosine levels and the progression of pulmonary fibrosis is critical for designing adenosine based approaches to treat pulmonary fibrosis. The goal of this study was to use various models of experimental lung fibrosis to understand when adenosine levels are elevated during pulmonary fibrosis and whether these elevations were associated with disease progression and severity. To accomplish this, extracellular adenosine levels, defined as adenosine levels found in bronchioalveolar lavage fluid, were determined in mouse models of resolvable and progressive pulmonary fibrosis. We found that relative bronchioalveolar lavage fluid adenosine levels are progressively elevated in association with pulmonary fibrosis and that adenosine levels diminish in association with the resolution of lung fibrosis. In addition, treatment of these models with dipyridamole, an inhibitor of nucleoside transporters that potentiates extracellular adenosine levels, demonstrated that the resolution of lung fibrosis is blocked by the failure of adenosine levels to subside. Furthermore, exacerbating adenosine levels led to worse fibrosis in a progressive fibrosis model. Increased adenosine levels were associated with elevation of IL-6 and IL-17, which are important inflammatory cytokines in pulmonary fibrosis. These results demonstrate that extracellular adenosine levels are closely associated with the progression of experimental pulmonary fibrosis and that this signaling pathway may mediate fibrosis by regulating IL-6 and IL-17 production. PMID:26527068

  3. Ticagrelor potentiates adenosine-induced stimulation of neutrophil chemotaxis and phagocytosis

    PubMed Central

    Alsharif, Khalaf F.; Thomas, Mark R.; Judge, Heather M.; Khan, Haroon; Prince, Lynne R.; Sabroe, Ian; Ridger, Victoria C.; Storey, Robert F.

    2015-01-01

    In the PLATO study, ticagrelor was associated with fewer pulmonary infections and subsequent deaths than clopidogrel. Neutrophils are a first-line defence against bacterial lung infection; ticagrelor inhibits cellular uptake of adenosine, a known regulator of neutrophil chemotaxis and phagocytosis. We assessed whether the inhibition of adenosine uptake by ticagrelor influences neutrophil chemotaxis and phagocytosis. Neutrophils and erythrocytes were isolated from healthy volunteers. Concentration-dependent effects of adenosine on IL-8-induced neutrophil chemotaxis were investigated and the involved receptors identified using adenosine receptor antagonists. The modulatory effects of ticagrelor on adenosine-mediated changes in neutrophil chemotaxis and phagocytosis of Streptococcus pneumoniae were determined in the presence of erythrocytes to replicate physiological conditions of cellular adenosine uptake. Low-concentration adenosine (10− 8 M) significantly increased IL-8-induced neutrophil chemotaxis (% neutrophil chemotaxis: adenosine 28.7% ± 4.4 vs. control 22.6% ± 2.4; p < 0.01) by acting on the high-affinity A1 receptor. Erythrocytes attenuated the effect of adenosine, although this was preserved by ticagrelor and dipyridamole (another inhibitor of adenosine uptake) but not by control or by cangrelor. Similarly, in the presence of erythrocytes, a low concentration of adenosine (10− 8 M) significantly increased neutrophil phagocytic index compared to control when ticagrelor was present (37.6 ± 6.6 vs. 28.0 ± 6.6; p = 0.028) but had no effect in the absence of ticagrelor. We therefore conclude that the inhibition of cellular adenosine reuptake by ticagrelor potentiates the effects of a nanomolar concentration of adenosine on neutrophil chemotaxis and phagocytosis. This represents a potential mechanism by which ticagrelor could influence host defence against bacterial lung infection. PMID:25869515

  4. Effects of adenosine metabolism in astrocytes on central nervous system oxygen toxicity.

    PubMed

    Chen, Yu-liang; Zhang, Ya-nan; Wang, Zhong-zhuang; Xu, Wei-gang; Li, Run-ping; Zhang, Jun-dong

    2016-03-15

    Hyperbaric oxygen (HBO) is widely used in military operations, especially underwater missions. However, prolonged and continuous inhalation of HBO can cause central nervous system oxygen toxicity (CNS-OT), which greatly limits HBO's application. The regulation of astrocytes to the metabolism of adenosine is involved in epilepsy. In our study, we aimed to observe the effects of HBO exposure on the metabolism of adenosine in the brain. Furthermore, we aimed to confirm the possible mechanism underlying adenosine's mediation of the CNS-OT. Firstly, anesthetized rats exposed to 5 atm absolute HBO for 80 min. The concentrations of extracellular adenosine, ATP, ADP, and AMP were detected. Secondly, free-moving rats were exposed to HBO at the same pressure for 20 min, and the activities of 5'-nucleotidase and ADK in brain tissues were measured. For the mechanism studies, we observed the effects of a series of different doses of drugs related to adenosine metabolism on the latency of CNS-OT. Results showed HBO exposure could increase adenosine content by inhibiting ADK activity and improving 5'-nucleotidase activity. And adenosine metabolism during HBO exposure may be a protective response against HBO-induced CNS-OT. Moreover, the improvement of adenosine concentration, activation of adenosine A1R, or suppression of ADK and adenosine A2AR, which are involved in the prevention of HBO-induced CNS-OT. This is the first study to demonstrate HBO exposure regulated adenosine metabolism in the brain. Adenosine metabolism and adenosine receptors are related to HBO-induced CNS-OT development. These results will provide new potential targets for the termination or the attenuation of CNS-OT. PMID:26806404

  5. Adenosine Signaling in Striatal Circuits and Alcohol Use Disorders

    PubMed Central

    Nam, Hyung Wook; Bruner, Robert C.; Choi, Doo-Sup

    2013-01-01

    Adenosine signaling has been implicated in the pathophysiology of alcohol use disorders and other psychiatric disorders such as anxiety and depression. Numerous studies have indicated a role for A1 receptors (A1R) in acute ethanol-induced motor incoordination, while A2A receptors (A2AR) mainly regulate the rewarding effect of ethanol in mice. Recent findings have demonstrated that dampened A2AR-mediated signaling in the dorsomedial striatum (DMS) promotes ethanol-seeking behaviors. Moreover, decreased A2AR function is associated with decreased CREB activity in the DMS, which enhances goal-oriented behaviors and contributes to excessive ethanol drinking in mice. Interestingly, caffeine, the most commonly used psychoactive substance, is known to inhibit both the A1R and A2AR. This dampened adenosine receptor function may mask some of the acute intoxicating effects of ethanol. Furthermore, based on the fact that A2AR activity plays a role in goal-directed behavior, caffeine may also promote ethanol-seeking behavior. The A2AR is enriched in the striatum and exclusively expressed in striatopallidal neurons, which may be responsible for the regulation of inhibitory behavioral control over drug rewarding processes through the indirect pathway of the basal ganglia circuit. Furthermore, the antagonistic interactions between adenosine and dopamine receptors in the striatum also play an integral role in alcoholism and addiction-related disorders. This review focuses on regulation of adenosine signaling in striatal circuits and the possible implication of caffeine in goal-directed behaviors and addiction. PMID:23912595

  6. Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise.

    PubMed

    Casey, Darren P; Madery, Brandon D; Pike, Tasha L; Eisenach, John H; Dietz, Niki M; Joyner, Michael J; Wilkins, Brad W

    2009-10-01

    We tested the hypothesis that adenosine contributes to augmented skeletal muscle vasodilation during hypoxic exercise. In separate protocols, subjects performed incremental rhythmic forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n = 8), subjects received an intra-arterial administration of saline (control) and aminophylline (adenosine receptor antagonist). In protocol 2 (n = 10), subjects received intra-arterial phentolamine (alpha-adrenoceptor antagonist) and combined phentolamine and aminophylline administration. Forearm vascular conductance (FVC; in ml x min(-1).100 mmHg(-1)) was calculated from forearm blood flow (in ml/min) and blood pressure (in mmHg). In protocol 1, the change in FVC (DeltaFVC; change from normoxic baseline) during hypoxic exercise with saline was 172 +/- 29 and 314 +/- 34 ml x min(-1) x 100 mmHg(-1) (10% and 20%, respectively). Aminophylline administration did not affect DeltaFVC during hypoxic exercise at 10% (190 +/- 29 ml x min(-1)x100 mmHg(-1), P = 0.4) or 20% (287 +/- 48 ml x min(-1) x 100 mmHg(-1), P = 0.3). In protocol 2, DeltaFVC due to hypoxic exercise with phentolamine infusion was 313 +/- 30 and 453 +/- 41 ml x min(-1) x 100 mmHg(-1) (10% and 20% respectively). DeltaFVC was similar at 10% (352 +/- 39 ml min(-1) x 100 mmHg(-1), P = 0.8) and 20% (528 +/- 45 ml x min(-1) x 100 mmHg(-1), P = 0.2) hypoxic exercise with combined phentolamine and aminophylline. In contrast, DeltaFVC to exogenous adenosine was reduced by aminophylline administration in both protocols (P < 0.05 for both). These observations suggest that adenosine receptor activation is not obligatory for the augmented hyperemia during hypoxic exercise in humans. PMID:19661449

  7. Adenosine signaling in striatal circuits and alcohol use disorders.

    PubMed

    Nam, Hyung Wook; Bruner, Robert C; Choi, Doo-Sup

    2013-09-01

    Adenosine signaling has been implicated in the pathophysiology of alcohol use disorders and other psychiatric disorders such as anxiety and depression. Numerous studies have indicated a role for A1 receptors (A1R) in acute ethanol-induced motor incoordination, while A2A receptors (A2AR) mainly regulate the rewarding effect of ethanol in mice. Recent findings have demonstrated that dampened A2AR-mediated signaling in the dorsomedial striatum (DMS) promotes ethanol-seeking behaviors. Moreover, decreased A2AR function is associated with decreased CREB activity in the DMS, which enhances goal-oriented behaviors and contributes to excessive ethanol drinking in mice. Interestingly, caffeine, the most commonly used psychoactive substance, is known to inhibit both the A1R and A2AR. This dampened adenosine receptor function may mask some of the acute intoxicating effects of ethanol. Furthermore, based on the fact that A2AR activity plays a role in goal-directed behavior, caffeine may also promote ethanol-seeking behavior. The A2AR is enriched in the striatum and exclusively expressed in striatopallidal neurons, which may be responsible for the regulation of inhibitory behavioral control over drug rewarding processes through the indirect pathway of the basal ganglia circuit. Furthermore, the antagonistic interactions between adenosine and dopamine receptors in the striatum also play an integral role in alcoholism and addiction-related disorders. This review focuses on regulation of adenosine signaling in striatal circuits and the possible implication of caffeine in goal-directed behaviors and addiction. PMID:23912595

  8. Anxiolytic activity of adenosine receptor activation in mice.

    PubMed

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P; Stone, T W

    1995-10-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  9. Anxiolytic activity of adenosine receptor activation in mice.

    PubMed Central

    Jain, N.; Kemp, N.; Adeyemo, O.; Buchanan, P.; Stone, T. W.

    1995-01-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  10. SwoHp, a Nucleoside Diphosphate Kinase, Is Essential in Aspergillus nidulans

    PubMed Central

    Lin, Xiaorong; Momany, Cory; Momany, Michelle

    2003-01-01

    The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ∼20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance. PMID:14665452

  11. Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

    PubMed

    Lian, Tiantian; Dong, Cai-Hong; Yang, Tao; Sun, Junde

    2014-01-01

    Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus. PMID:24941033

  12. The antihyperalgesic effect of cytidine-5'-diphosphate-choline in neuropathic and inflammatory pain models.

    PubMed

    Bagdas, Deniz; Sonat, Fusun Ak; Hamurtekin, Emre; Sonal, Songul; Gurun, Mine Sibel

    2011-09-01

    This study was designed to test the effects of intracerebroventricularly (i.c.v.) administered CDP-choline (cytidine-5'-diphosphate-choline; citicoline) and its metabolites in rat models of inflammatory and neuropathic pain. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 µmol) produced a dose and time-dependent reversal of mechanical hyperalgesia in both carrageenan-induced inflammatory and chronic constriction injury-induced neuropathic pain models in rats. The antihyperalgesic effect of CDP-choline was similar to that observed with an equimolar dose of choline (1 µmol). The CDP-choline-induced antihyperalgesic effect was prevented by central administration of the neuronal high-affinity choline uptake inhibitor hemicholinium-3 (1 µg), the nonselective nicotinic receptor antagonist mecamylamine (50 µg), the α7-selective nicotinic ACh receptor antagonist, α-bungarotoxin (2 µg) and the γ-aminobutyric acid B receptor antagonist CGP-35348 (20 µg). In contrast, i.c.v. pretreatment with the nonselective opioid receptor antagonist naloxone (10 µg) only prevented the CDP-choline-induced antihyperalgesic effect in the neuropathic pain model while the nonselective muscarinic receptor antagonist atropine (10 µg) did not alter the antihyperalgesic effect in the two models. These results indicate that CDP-choline-elicited antihyperalgesic effect in different models of pain occurs through mechanisms that seem to involve an interaction with supraspinal α7-selective nicotinic ACh receptors, and γ-aminobutyric acid B receptors, whereas central opioid receptors have a role only in the neuropathic pain model. PMID:21836465

  13. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

    PubMed

    Goubau, Delphine; Schlee, Martin; Deddouche, Safia; Pruijssers, Andrea J; Zillinger, Thomas; Goldeck, Marion; Schuberth, Christine; Van der Veen, Annemarthe G; Fujimura, Tsutomu; Rehwinkel, Jan; Iskarpatyoti, Jason A; Barchet, Winfried; Ludwig, Janos; Dermody, Terence S; Hartmann, Gunther; Reis e Sousa, Caetano

    2014-10-16

    Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system. PMID:25119032

  14. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates

    PubMed Central

    Deddouche, Safia; Pruijssers, Andrea J.; Zillinger, Thomas; Goldeck, Marion; Schuberth, Christine; Van der Veen, Annemarthe G.; Fujimura, Tsutomu; Rehwinkel, Jan; Iskarpatyoti, Jason A.; Barchet, Winfried; Ludwig, Janos; Dermody, Terence S.; Hartmann, Gunther; Reis e Sousa, Caetano

    2014-01-01

    SUMMARY Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection1,2. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α/β; hereafter IFN), key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5′-end (reviewed in 1-3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5′-diphosphates (5′pp). Genomes from mammalian reoviruses with 5′pp termini, 5′pp-RNA isolated from yeast L-A virus, and base-paired 5′pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5′pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5′pp. Such RNAs are found in some viruses but not uninfected cells, indicating that recognition of 5′pp-RNA, like that of 5′ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system. PMID:25119032

  15. A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

    PubMed Central

    Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Tamada, Taro; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

    2012-01-01

    Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography. The molecular weight of HaNDK is 33,660, and the X-ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X-ray crystallography to 2.3 and 2.5 Å resolution with space groups of P21 and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild-type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances. PMID:22275000

  16. Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase, an enzyme of isopentenyl diphosphate biosynthesis.

    PubMed

    Sutherlin, Autumn; Hedl, Matija; Sanchez-Neri, Barbara; Burgner, John W; Stauffacher, Cynthia V; Rodwell, Victor W

    2002-08-01

    Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis. PMID:12107122

  17. Inhibition of chemically-induced neoplastic transformation by a novel tetrasodium diphosphate astaxanthin derivative.

    PubMed

    Hix, Laura M; Frey, Dean A; McLaws, Mark D; Østerlie, Marianne; Lockwood, Samuel F; Bertram, John S

    2005-09-01

    Carotenoids have been implicated in numerous epidemiological studies as being protective against cancer at many sites, and their chemopreventive properties have been confirmed in laboratory studies. Astaxanthin (AST), primarily a carotenoid of marine origin, responsible for the pink coloration of salmon, shrimp and lobster, has received relatively little attention. As with other carotenoids, its highly lipophilic properties complicate delivery to model systems. To overcome this issue we have synthesized a novel tetrasodium diphosphate astaxanthin (pAST) derivative with aqueous dispersibility of 25.21 mg/ml. pAST was delivered to C3H/10T1/2 cells in an aqueous/ethanol solution and compared with non-esterified AST dissolved in tetrahydrofuran. We show pAST to (i) upregulate connexin 43 (Cx43) protein expression; (ii) increase the formation of Cx43 immunoreactive plaques; (iii) upregulate gap junctional intercellular communication (GJIC); and (iv) cause 100% inhibition of methylcholanthrene-induced neoplastic transformation at 10(-6) M. In all these assays, pAST was superior to non-esterified AST itself; in fact, pAST exceeded the potency of all other previously tested carotenoids in this model system. Cleavage of pAST to non-esterified (free) AST and uptake into cells was also verified by HPLC; however, levels of free AST were approximately 100-fold lower than in cells treated with AST itself, suggesting that pAST possesses intrinsic activity. The dual properties of water dispersibility (enabling parenteral administration in vivo) and increased potency should prove extremely useful in the future development of cancer chemopreventive agents. PMID:15888493

  18. Uridine Diphosphate-Glucuronosyltransferase (UGT) Xenobiotic Metabolizing Activity and Genetic Evolution in Pinniped Species.

    PubMed

    Kakehi, Mayu; Ikenaka, Yoshinori; Nakayama, Shouta M M; Kawai, Yusuke K; Watanabe, Kensuke P; Mizukawa, Hazuki; Nomiyama, Kei; Tanabe, Shinsuke; Ishizuka, Mayumi

    2015-10-01

    There are various interspecies differences in xenobiotic-metabolizing enzymes. It is known that cats show slow glucuronidation of drugs such as acetaminophen and strong side effects due to the UGT1A6 pseudogene. Recently, the UGT1A6 pseudogene was found in the Northern elephant seal and Otariidae was suggested to be UGT1A6-deficient. From the results of measurements of uridine diphosphate-glucuronosyltransferase (UGT) activity using liver microsomes, the Steller sea lion, Northern fur seal, and Caspian seal showed UGT activity toward 1-hydroxypyrene and acetaminophen as low as in cats, which was significantly lower than in rat and dog. Furthermore, UGT1A6 pseudogenes were found in Steller sea lion and Northern fur seal, and all Otariidae species were suggested to have the UGT1A6 pseudogene. The UGT1 family genes appear to have undergone birth-and-death evolution based on a phylogenetic and synteny analysis of the UGT1 family in mammals including Carnivora. UGT1A2-1A5 and UGT1A7-1A10 are paralogous genes to UGT1A1 and UGTA6, respectively, and their numbers were lower in cat, ferret and Pacific walrus than in human, rat, and dog. Felidae and Pinnipedia, which are less exposed to natural xenobiotics such as plant-derived toxins due to their carnivorous diet, have experienced fewer gene duplications of xenobiotic-metabolizing UGT genes, and even possess UGT1A6 pseudogenes. Artificial environmental pollutants and drugs conjugated by UGT are increasing dramatically, and their elimination to the environment can be of great consequence to cat and Pinnipedia species, whose low xenobiotic glucuronidation capacity makes them highly sensitive to these compounds. PMID:26179383

  19. Molecular cloning and characterization of three isoprenyl diphosphate synthase genes from alfalfa.

    PubMed

    Sun, Yan; Long, Ruicai; Kang, Junmei; Zhang, Tiejun; Zhang, Ze; Zhou, He; Yang, Qingchuan

    2013-02-01

    Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid, gibberellins, chlorophylls and many other products in plants. Saponins are an important group of bioactive plant natural products. The alfalfa (Medicago sativa L.) saponins are glycosides of different triterpene aglycones and possess many biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS) encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS expressed in Escherichia coli indicated that they all had IDS activity. Expression analysis of the three genes in different alfalfa tissues using real time PCR displayed that they were expressed in all tissues although they had a different expression patterns. MsFPPS and MsGPS displayed a significant increase in transcript level in response to methyl jasmonate, but the transcript level of MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS showed high levels of squalene content when compared with control. However, no differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In addition, the overexpression of MsFPPS induced senescence response in transgenic plant leaves. This result may indicate that MsFPPS performs a role not only in phytosterol and triterpene biosynthesis, but also in growth regulation. PMID:23238915

  20. Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

    PubMed

    Hasnain, Ghulam; Roje, Sanja; Sa, Na; Zallot, Rémi; Ziemak, Michael J; de Crécy-Lagard, Valérie; Gregory, Jesse F; Hanson, Andrew D

    2016-01-15

    The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family. PMID:26537753

  1. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  2. METABOLIC REGULATION OF ADENOSINE TRIPHOSPHATE SULFURYLASE IN YEAST

    PubMed Central

    de Vito, Peter C.; Dreyfuss, Jacques

    1964-01-01

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

  3. Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

    PubMed Central

    Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

    2014-01-01

    D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury. PMID:24407238

  4. Genetic removal of the A2A adenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice.

    PubMed

    Mohsenin, Amir; Mi, Tiejuan; Xia, Yang; Kellems, Rodney E; Chen, Jiang-Fan; Blackburn, Michael R

    2007-09-01

    Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A(2A) adenosine receptor (A(2A)R) functions to limit inflammation and promote tissue protection; however, the contribution of A(2A)R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A(2A)R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A(2A)R double knockout mice. Genetic removal of the A(2A)R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A(2A)R. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-alpha and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A(2A)R double knockout mice. These findings suggest that the A(2A)R plays a protective role in the ADA-deficient model of pulmonary inflammation. PMID:17601796

  5. Effect in vitro on platelet function of two compounds developed from the pyrimido-pyrimidines

    PubMed Central

    Slater, S. D.; Turpie, A. G. G.; Douglas, A. S.; McNicol, G. P.

    1972-01-01

    VK 774 and VK 744, two new compounds developed from the pyrimido-pyrimidines, have been found to be powerful inhibitors of platelet function tested in vitro. They inhibit adenosine diphosphate (ADP)-induced platelet aggregation, and the release of platelet factor 3 by kaolin, and VK 774 also reduces platelet adhesiveness and inhibits platelet aggregation (`snowstorm' effect) in the Chandler tube system. Although measured percentage whole blood clot retraction was uninfluenced by these drugs the clot produced with VK 774 was friable and soft. VK 774 appears to be the most powerful of these compounds reported so far, being active in some test systems at 10−6M, and, if the results of toxicity testing are satisfactory, it should be an important agent for therapeutic trial. PMID:5046075

  6. [Effects of dopamine and adenosine on regulation of water-electrolyte exchange in Amoeba proteus].

    PubMed

    Bagrov, Ia Iu; Manusova, N B

    2014-01-01

    Dopamine and adenosine both regulate transport of sodium chloride in the renal tubules in mammals. We have studied the effect of dopamine and adenosine on spontaneous activity of contractile vacuole of Amoeba proteous. Both substances stimulated contractile vacuole. The effect of dopamine was suppressed by D2 receptor antagonist, haloperidol, but not by D1 antagonist, SCH 39166. Adenylate cyclase inhibitor, 2.5-dideoxyadenosine, suppressed the effect of dopamine, but not of adenosine. Inhibitor of protein kinase C, staurosporine, in contrast, blocked the effect of adenosine, but not dopamine. Notably, dopamine opposed effect of adenosine and vice versa. These results suggest that similar effects of dopamine and adenosine could be mediated by different intracellulare mechanisms. PMID:25509166

  7. Sustained Elevated Adenosine via ADORA2B Promotes Chronic Pain through Neuro-immune Interaction.

    PubMed

    Hu, Xia; Adebiyi, Morayo G; Luo, Jialie; Sun, Kaiqi; Le, Thanh-Thuy T; Zhang, Yujin; Wu, Hongyu; Zhao, Shushan; Karmouty-Quintana, Harry; Liu, Hong; Huang, Aji; Wen, Yuan Edward; Zaika, Oleg L; Mamenko, Mykola; Pochynyuk, Oleh M; Kellems, Rodney E; Eltzschig, Holger K; Blackburn, Michael R; Walters, Edgar T; Huang, Dong; Hu, Hongzhen; Xia, Yang

    2016-06-28

    The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada(-/-) mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund's adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets. PMID:27320922

  8. 2-Triazole-Substituted Adenosines: A New Class of Selective A3 Adenosine Receptor Agonists, Partial Agonists, and Antagonists

    PubMed Central

    Cosyn, Liesbet; Palaniappan, Krishnan K.; Kim, Soo-Kyung; Duong, Heng T.; Gao, Zhan-Guo; Jacobson, Kenneth A.; Van Calenbergh, Serge

    2016-01-01

    “Click chemistry” was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1–14). Binding affinity at the human A1, A2A, and A3ARs (adenosine receptors) and relative efficacy at the A3AR were determined. Some triazol-1-yl analogues showed A3AR affinity in the low nanomolar range, a high ratio of A3/A2A selectivity, and a moderate-to-high A3/A1 ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A3AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A3AR efficacy. Thus, several 5′-OH derivatives appeared to be selective A3AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A1AR and displaying a characteristic docking mode in an A3AR model. The corresponding 5′-ethyluronamide analogues generally showed increased A3AR affinity and behaved as full agonists, i.e., 17, with 910-fold A3/A1 selectivity. Thus, N6-substituted 2-(1,2,3-triazolyl)-adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A3AR antagonists, partial agonists, and agonists. PMID:17149867

  9. Overexpression of an Isoprenyl Diphosphate Synthase in Spruce Leads to Unexpected Terpene Diversion Products That Function in Plant Defense1[W][OPEN

    PubMed Central

    Nagel, Raimund; Berasategui, Aileen; Paetz, Christian; Gershenzon, Jonathan; Schmidt, Axel

    2014-01-01

    Spruce (Picea spp.) and other conifers employ terpenoid-based oleoresin as part of their defense against herbivores and pathogens. The short-chain isoprenyl diphosphate synthases (IDS) are situated at critical branch points in terpene biosynthesis, producing the precursors of the different terpenoid classes. To determine the role of IDS and to create altered terpene phenotypes for assessing the defensive role of terpenoids, we overexpressed a bifunctional spruce IDS, a geranyl diphosphate and geranylgeranyl diphosphate synthase in white spruce (Picea glauca) saplings. While transcript level (350-fold), enzyme activity level (7-fold), and in planta geranyl diphosphate and geranylgeranyl diphosphate levels (4- to 8-fold) were significantly increased in the needles of transgenic plants, there was no increase in the major monoterpenes and diterpene acids of the resin and no change in primary isoprenoids, such as sterols, chlorophylls, and carotenoids. Instead, large amounts of geranylgeranyl fatty acid esters, known from various gymnosperm and angiosperm plant species, accumulated in needles and were shown to act defensively in reducing the performance of larvae of the nun moth (Lymantria monacha), a conifer pest in Eurasia. These results show the impact of overexpression of an IDS and the defensive role of an unexpected accumulation product of terpenoid biosynthesis with the potential for a broader function in plant protection. PMID:24346420

  10. PAP and NT5E inhibit nociceptive neurotransmission by rapidly hydrolyzing nucleotides to adenosine

    PubMed Central

    2011-01-01

    Background Prostatic acid phosphatase (PAP) and ecto-5'-nucleotidase (NT5E, CD73) produce extracellular adenosine from the nucleotide AMP in spinal nociceptive (pain-sensing) circuits; however, it is currently unknown if these are the main ectonucleotidases that generate adenosine or how rapidly they generate adenosine. Results We found that AMP hydrolysis, when measured histochemically, was nearly abolished in dorsal root ganglia (DRG) neurons and lamina II of spinal cord from Pap/Nt5e double knockout (dKO) mice. Likewise, the antinociceptive effects of AMP, when combined with nucleoside transport inhibitors (dipyridamole or 5-iodotubericidin), were reduced by 80-100% in dKO mice. In addition, we used fast scan cyclic voltammetry (FSCV) to measure adenosine production at subsecond resolution within lamina II. Adenosine was maximally produced within seconds from AMP in wild-type (WT) mice but production was reduced >50% in dKO mice, indicating PAP and NT5E rapidly generate adenosine in lamina II. Unexpectedly, we also detected spontaneous low frequency adenosine transients in lamina II with FSCV. Adenosine transients were of short duration (<2 s) and were reduced (>60%) in frequency in Pap-/-, Nt5e-/- and dKO mice, suggesting these ectonucleotidases rapidly hydrolyze endogenously released nucleotides to adenosine. Field potential recordings in lamina II and behavioral studies indicate that adenosine made by these enzymes acts through the adenosine A1 receptor to inhibit excitatory neurotransmission and nociception. Conclusions Collectively, our experiments indicate that PAP and NT5E are the main ectonucleotidases that generate adenosine in nociceptive circuits and indicate these enzymes transform pulsatile or sustained nucleotide release into an inhibitory adenosinergic signal. PMID:22011440

  11. Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons.

    PubMed

    Liu, Tao; Fujita, Tsugumi; Kawasaki, Yasuhiko; Kumamoto, Eiichi

    2004-07-30

    A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs. PMID:15275960

  12. Estimation of skeletal muscle interstitial adenosine during forearm dynamic exercise in humans

    NASA Technical Reports Server (NTRS)

    Costa, F.; Heusinkveld, J.; Ballog, R.; Davis, S.; Biaggioni, I.

    2000-01-01

    It has been proposed that adenosine is a metabolic signal that triggers activation of muscle afferents involved in the exercise pressor reflex. Furthermore, exogenous adenosine induces sympathetic activation that mimics the exercise pressor reflex, and blockade of adenosine receptors inhibits sympathetic activation induced by exercise. Thus, we hypothesize that adenosine is released locally by the muscle during exercise. We used microdialysis probes, placed in the flexor digitorium superficialis muscle, to estimate muscle interstitial adenosine levels in humans. We estimated resting in vivo muscle interstitial adenosine concentrations (0.292+/-0.058 micromol/L, n=4) by perfusing increasing concentrations of adenosine to determine the gradient produced in the dialysate. Muscle interstitial adenosine concentrations increased from 0.23+/-0.04 to 0.82+/-0.14 micromol/L (n=14, P<0.001) during intermittent dynamic exercise at 50% of maximal voluntary contraction. Lactate increased from 0.8+/-0.1 to 2.3+/-0.3 mmol/L (P<0.001). Lower intensity (15% maximal voluntary contraction) intermittent dynamic exercise increased adenosine concentrations from 0.104+/-0.02 to 0.42+/-0.16 micromol/L (n=7). The addition of ischemia to this low level of exercise produced a greater increase in adenosine (from 0.095+/-0.02 to 0.48+/-0.2 micromol/L) compared with nonischemic exercise (0. 095+/-0.02 to 0.25+/-0.12 micromol/L). These results indicate that microdialysis is useful in estimating adenosine concentrations and in reflecting changes in muscle interstitial adenosine during dynamic exercise in humans.

  13. Biosynthesis of Taxadiene in Saccharomyces cerevisiae : selection of geranylgeranyl diphosphate synthase directed by a computer-aided docking strategy.

    PubMed

    Ding, Ming-Zhu; Yan, Hui-Fang; Li, Lin-Feng; Zhai, Fang; Shang, Lu-Qing; Yin, Zheng; Yuan, Ying-Jin

    2014-01-01

    Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells. PMID:25295588

  14. Biosynthesis of Taxadiene in Saccharomyces cerevisiae : Selection of Geranylgeranyl Diphosphate Synthase Directed by a Computer-Aided Docking Strategy

    PubMed Central

    Li, Lin-feng; Zhai, Fang; Shang, Lu-qing; Yin, Zheng; Yuan, Ying-jin

    2014-01-01

    Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1st, 4th and 6th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells. PMID:25295588

  15. Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion.

    PubMed Central

    Diefenbach, R J; Duggleby, R G

    1991-01-01

    To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit. PMID:2049073

  16. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    SciTech Connect

    Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric; Kaiser, Marcel; Amzel, L. Mario; Gabelli, Sandra B.

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  17. Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

    PubMed

    Misra, Rajesh Chandra; Garg, Anchal; Roy, Sudeep; Chanotiya, Chandan Singh; Vasudev, Prema G; Ghosh, Sumit

    2015-11-01

    Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance. PMID:26475187

  18. Kinetic and thermodynamic studies of the dissolution of thorium-uranium (IV) phosphate-diphosphate solid solutions

    NASA Astrophysics Data System (ADS)

    Thomas, A. C.; Dacheux, N.; Le Coustumer, P.; Brandel, V.; Genet, M.

    2001-06-01

    The dissolution of thorium-uranium (IV) phosphate-diphosphate solid solutions (TUPD) was studied as a function of the temperature and leachate acidity. The dependence of the normalized dissolution rate on the temperature leads to an activation energy equal to about 40 kJ mol -1, close to that obtained for the pure thorium phosphate-diphosphate ( 42±3 kJ mol-1) and for thorium-plutonium (IV) phosphate-diphosphate solid solutions ( 41±1 kJ mol-1). The normalized dissolution rate of TUPD slightly increases with the leachate acidity. The partial order related to the proton concentration, n, is equal to 0.40±0.02 while the apparent normalized dissolution rate constant, k'T,I, reaches (2.8±0.7)×10 -4 g m-2 d-1 at 90°C and for [ H3O+]=1 M. When the saturation of the leachate is reached, the concentration of thorium, uranium and phosphate ions measured in the solution are controlled by the precipitation of the uranyl phosphate pentahydrate (UO 2) 3(PO 4) 2·5H 2O and the thorium phosphate-hydrogenphosphate Th 2(PO 4) 2(HPO 4)·H 2O. Both solids were extensively characterized using XRD, infrared and UV-visible spectroscopies or electron probe microanalysis (EPMA). Their solubility products, K°S,0, were determined and extrapolated to I=0. They are equal to 10 -55.2±0.5 and 10 -66.6±1.2, respectively. All the samples leached were characterized using EPMA, SEM and TEM. These techniques showed that during the dissolution process, thorium and uranium are completely separated as (UO 2) 3(PO 4) 2·5H 2O, on one hand, and Th 2(PO 4) 2(HPO 4)·H 2O, on the other hand. In the first days of leaching tests, an amorphous additional phase, identified as Th 2(PO 4) 2(HPO 4)· nH 2O was also observed. Several leaching tests performed on sintered TUPD samples revealed that the dissolution rates measured in 10 -1 M HNO3 is very low (6.5×10 -5 g d-1) by comparison to other ceramics studied in the same objective. In these conditions, the thorium phosphate-diphosphate (TPD) appears as

  19. Fructose-1,6-diphosphate as an in vitro and in vivo anti-alcohol agent in the rat.

    PubMed

    Galzigna, L; Bianchi, M; Rizzoli, V; Scuri, R; Giannetti, P; Paesano, A

    1990-01-01

    Fructose-1, 6-diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol-stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol-stimulated [36Cl-]-uptake by rat brain microvesicles and affects the isolated GABA-receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose-dependent decrease in ethanol-induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP. PMID:2160337

  20. A tailor-made chimeric thiamine diphosphate dependent enzyme for the direct asymmetric synthesis of (S)-benzoins.

    PubMed

    Westphal, Robert; Vogel, Constantin; Schmitz, Carlo; Pleiss, Jürgen; Müller, Michael; Pohl, Martina; Rother, Dörte

    2014-08-25

    Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion. PMID:25044968

  1. Synthesis and biological evaluation of clitocine analogues as adenosine kinase inhibitors.

    PubMed

    Lee, C H; Daanen, J F; Jiang, M; Yu, H; Kohlhaas, K L; Alexander, K; Jarvis, M F; Kowaluk, E L; Bhagwat, S S

    2001-09-17

    Adenosine kinase (AK) is the primary enzyme responsible for adenosine metabolism. Inhibition of AK effectively increases extracellular adenosine concentrations and represents an alternative approach to enhance the beneficial actions of adenosine as compared to direct-acting receptor agonists. Clitocine (3), isolated from the mushroom Clitocybe inversa, has been found to be a weak inhibitor of AK. We have prepared a number of analogues of clitocine in order to improve its potency and demonstrated that 5'-deoxy-5'-amino-clitocine (7) improved AK inhibitory potency by 50-fold. PMID:11549437

  2. Characterization and regulation of adenosine transport in T84 intestinal epithelial cells.

    PubMed

    Mun, E C; Tally, K J; Matthews, J B

    1998-02-01

    Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl- secretion. Previous data suggest that A2b receptor-mediated Cl- secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na(+)-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5'-(N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl- secretory responses and hence diarrheal disorders. PMID:9486178

  3. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

  4. Molecular vibration-activity relationship in the agonism of adenosine receptors.

    PubMed

    Chee, Hyun Keun; Oh, S June

    2013-12-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  5. Adenosine A2A receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC

    PubMed Central

    Brand, Frank; Klutz, Athena; Jacobson, Kenneth A.; Fredholm, Bertil B.; Schulte, Gunnar

    2009-01-01

    G protein-coupled receptors, such as the adenosine A2A receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A2A receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A2A (Ki = 149 ± 27 nM) as well as A3 receptors (Ki= 240 ± 160 nM) but not to adenosine A1 receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand’s functionality at adenosine A2A but not A2B receptors. In live cell imaging studies, Alexa488-APEC induced adenosine A2A receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A2A receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A2A receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC provided here showed that it provides a usefultool for tracing adenosine A2A receptors in vitro. PMID:18603240

  6. Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

    PubMed Central

    Kelley, G G; Aassar, O S; Forrest, J N

    1991-01-01

    The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport. Images PMID:1752953

  7. Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism.

    PubMed

    Nguyen, Michael D; Ross, Ashley E; Ryals, Matthew; Lee, Scott T; Venton, B Jill

    2015-12-01

    Adenosine is a neuromodulator that regulates neurotransmission in the brain and central nervous system. Recently, spontaneous adenosine release that is cleared in 3-4 sec was discovered in mouse spinal cord slices and anesthetized rat brains. Here, we examined the clearance of spontaneous adenosine in the rat caudate-putamen and exogenously applied adenosine in caudate brain slices. The V max for clearance of exogenously applied adenosine in brain slices was 1.4 ± 0.1 μmol/L/sec. In vivo, the equilibrative nucleoside transport 1 (ENT1) inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI) (1 mg/kg, i.p.) significantly increased the duration of adenosine, while the ENT1/2 inhibitor, dipyridamole (10 mg/kg, i.p.), did not affect duration. 5-(3-Bromophenyl)-7-[6-(4-morpholinyl)-3-pyrido[2,3-d]byrimidin-4-amine dihydrochloride (ABT-702), an adenosine kinase inhibitor (5 mg/kg, i.p.), increased the duration of spontaneous adenosine release. The adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (10 mg/kg, i.p.), also increased the duration in vivo. Similarly, NBTI (10 μmol/L), ABT-702 (100 nmol/L), or EHNA (20 μmol/L) also decreased the clearance rate of exogenously applied adenosine in brain slices. The increases in duration for blocking ENT1, adenosine kinase, or adenosine deaminase individually were similar, about 0.4 sec in vivo; thus, the removal of adenosine on a rapid time scale occurs through three mechanisms that have comparable effects. A cocktail of ABT-702, NBTI, and EHNA significantly increased the duration by 0.7 sec, so the mechanisms are not additive and there may be additional mechanisms clearing adenosine on a rapid time scale. The presence of multiple mechanisms for adenosine clearance on a time scale of seconds demonstrates that adenosine is tightly regulated in the extracellular space. PMID:27022463

  8. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  9. Modulation of adenosine signaling prevents scopolamine-induced cognitive impairment in zebrafish.

    PubMed

    Bortolotto, Josiane Woutheres; Melo, Gabriela Madalena de; Cognato, Giana de Paula; Vianna, Mônica Ryff Moreira; Bonan, Carla Denise

    2015-02-01

    Adenosine, a purine ribonucleoside, exhibits neuromodulatory and neuroprotective effects in the brain and is involved in memory formation and cognitive function. Adenosine signaling is mediated by adenosine receptors (A1, A2A, A2B, and A3); in turn, nucleotide and nucleoside-metabolizing enzymes and adenosine transporters regulate its levels. Scopolamine, a muscarinic cholinergic receptor antagonist, has profound amnesic effects in a variety of learning paradigms and has been used to induce cognitive deficits in animal models. This study investigated the effects of acute exposure to caffeine (a non-selective antagonist of adenosine receptors A1 and A2A), ZM 241385 (adenosine receptor A2A antagonist), DPCPX (adenosine receptor A1 antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in a model of pharmacological cognitive impairment induced by scopolamine in adult zebrafish. Caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered independently via i.p. in zebrafish, followed by exposure to scopolamine dissolved in tank water (200μM). These compounds prevented the scopolamine-induced amnesia without impacting locomotor activity or social interaction. Together, these data support the hypothesis that adenosine signaling may modulate memory processing, suggesting that these compounds present a potential preventive strategy against cognitive impairment. PMID:25490060

  10. Adenosine-induced coronary vasospasm following drug-eluting stent implantation

    PubMed Central

    Matsumoto, Naoya; Nagao, Ken; Hirayama, Atsushi; Kasama, Shu

    2014-01-01

    We present the case of coronary vasospasm during adenosine stress in a patient with a prior drug-eluting stent implantation. The patient had a stent implantation in the left anterior descending coronary artery 3 years ago. Recently, he developed a chest pain and underwent adenosine stress myocardial perfusion single photon emission CT (SPECT). During the adenosine stress, he felt severe chest pain and ST elevation on electrocardiogram. An invasive coronary angiography showed no in-stent restenosis. This phenomenon deemed to be adenosine-induced coronary vasospasm after stent implantation. PMID:24518394

  11. Effects of adenosine and adenosine A2A receptor agonist on motor nerve conduction velocity and nerve blood flow in experimental diabetic neuropathy.

    PubMed

    Kumar, Sokindra; Arun, K H S; Kaul, Chaman L; Sharma, Shyam S

    2005-01-01

    This study examined the effects of chronic administration of adenosine and CGS 21680 hydrochloride (adenosine A(2A) receptor agonist) on motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and histology of sciatic nerve in animal model of diabetic neuropathy. Adenosinergic agents were administered for 2 weeks after 6 weeks of streptozotocin-induced (50 mg/kg i.p.) diabetes in male Sprague-Dawley rats. Significant reduction in sciatic MNCV and NBF were observed after 8 weeks in diabetic animals in comparison with control (non diabetic) rats. Adenosine (10 mg/kg, i.p.) significantly improved sciatic MNCV and NBF in diabetic rats. The protective effect of adenosine on MNCV and NBF was completely reversed by theophylline (50 mg/kg, i.p.), a non-selective adenosine receptor antagonist, suggesting that the adenosine effect was mediated via adenosinergic receptors. CGS 21680 (0.1 mg/kg, i.p.) significantly improved NBF; however, MNCV was not significantly improved in diabetic rats. At a dose of 1 mg/kg, neither MNCV nor NBF was improved by CGS 21680 in diabetic rats. ZM 241385 (adenosine A(2A) receptor antagonist) prevented the effect of CGS 21680 (0.1 mg/kg, i.p.). Histological changes observed in sciatic nerve were partially improved by the adenosinergic agents in diabetic rats. Results of the present study, suggest the potential of adenosinergic agents in the therapy of diabetic neuropathy. PMID:15829161

  12. Sex Hormones Regulate Tenofovir-Diphosphate in Female Reproductive Tract Cells in Culture

    PubMed Central

    Shen, Zheng; Fahey, John V.; Bodwell, Jack E.; Rodriguez-Garcia, Marta; Kashuba, Angela D. M.; Wira, Charles R.

    2014-01-01

    The conflicting results of recent pre-exposure prophylaxis (PrEP) trials utilizing tenofovir (TFV) to prevent HIV infection in women led us to evaluate the accumulation of intracellular TFV-diphosphate (TFV-DP) in cells from the female reproductive tract (FRT) and whether sex hormones influence the presence of TFV-DP in these cells. Following incubation with TFV, isolated epithelial cells, fibroblasts, CD4+ T cells and CD14+ cells from the FRT as well as blood CD4+ T cells and monocyte-derived macrophages convert TFV to TFV-DP. Unexpectedly, we found that TFV-DP concentrations (fmol/million cells) vary significantly with the cell type analyzed and the site within the FRT. Epithelial cells had 5-fold higher TFV-DP concentrations than fibroblasts; endometrial epithelial cells had higher TFV-DP concentrations than cells from the ectocervix. Epithelial cells had 125-fold higher TFV-DP concentrations than FRT CD4+ T cells, which were comparable to that measured in peripheral blood CD4+ T cells. These findings suggest the existence of a TFV-DP gradient in the FRT where epithelial cells > fibroblasts > CD4+ T cells and macrophages. In other studies, estradiol increased TFV-DP concentrations in endometrial and endocervical/ectocervical epithelial cells, but had no effect on fibroblasts or CD4+ T cells from FRT tissues. In contrast, progesterone alone and in combination with estradiol decreased TFV-DP concentrations in FRT CD4+ T cells. Our results suggest that epithelial cells and fibroblasts are a repository of TFV-DP that is under hormonal control. These cells might act either as a sink to decrease TFV availability to CD4+ T cells and macrophages in the FRT, or upon conversion of TFV-DP to TFV increase TFV availability to HIV-target cells. In summary, these results indicate that intracellular TFV-DP varies with cell type and location in the FRT and demonstrate that estradiol and/or progesterone regulate the intracellular concentrations of TFV-DP in FRT epithelial cells

  13. Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells.

    PubMed

    Ehringer, W D; Niu, W; Chiang, B; Wang, O L; Gordon, L; Chien, S

    2000-07-01

    Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-alpha-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of 14C-FDP by endothelial cells cultured under hypoxia

  14. Successful treatment of irreversible hemorrhagic shock in dogs with fructose-1,6 diphosphate and dichloroacetate.

    PubMed

    Granot, H; Steiner, I

    1985-01-01

    Hemodynamic and metabolic effects of fructose-1,6-diphosphate (F.D.P.) and dichloroacetate sodium (D.C.A.) administration were studied in 17 mongrel dogs during experimentally induced hemorrhagic shock using a modified Wigger's technique. During the oligemic period, which was maintained for 3 hours, a control group of animals (A) received a 5% glucose solution at a rate of 3 mg/kg/min, while the treated group (B) received D.C.A. (175 mg/kg for 30 minutes) and F.D.P. (5 mg/kg/min) as aqueous solutions. After retransfusion of the shed blood, both groups of animals were left to recover. All eight dogs of the control group died within 3 hours following the experiment, while six out of the nine treated dogs survived during a week of follow-up (p = 0.007). Two hours after retransfusion, blood pressure and cardiac index in group B returned to control levels (115 +/- 4.8 mmHg and 0.097 +/- 0.008 liters/min/kg), while group A demonstrated a rapid and progressive deterioration (64 +/- 9.7 mmHg and 0.041 +/- 0.005 liters/min/kg). Severe core hypothermia (down to 33.3 degrees C) developed in group A dogs despite retransfusion, while a normal core temperature was maintained in the treated dogs. Calculated oxygen consumption during the oligemic period was significantly higher in group B animals despite similar calculated oxygen delivery in both groups of animals. Hyperlactemia was significantly lower in group B animals despite F.D.P. administration. This can be attributed to the addition of D.C.A. to the treatment. F.D.P. and D.C.A. administration prevented the occurrence of respiratory failure resulting, most probably, from respiratory muscle fatigue owing to depressed metabolic rate and increased lactate formation in these muscles during the shock period. It is suggested that administration of F.D.P. and D.C.A. during hemorrhagic shock in dogs has a favorable effect on the outcome of this life-threatening condition. PMID:3978767

  15. An industrial process for selective synthesis of 7-methyl guanosine 5'-diphosphate: versatile synthon for synthesis of mRNA cap analogues.

    PubMed

    Kore, Anilkumar R; Parmar, Gaurang

    2006-03-01

    We report an industrial scale facile synthesis of 7-methyl guanosine 5'-diphosphate, which plays an important role in synthesis of various mRNA cap analogs. An efficient and selective methylation at position 7 of guanosine 5'-diphosphate was achieved by dissolving guanosine 5'-diphosphate in water and drops wise addition of dimethyl sulfate over a period of 1 h at room temperature. The reaction was completed within 2 h and resulted in more than a 96% yield. The desired product, 7-methyl GDP was purified by using BPG column on AKTA Purifier 100. Certainly, this method has advantages over the known methylation method, in terms of yield, economy, safety, and environmental concerns. PMID:16629126

  16. The occurrence of uridine diphosphate N-acetylgalactosamine 6-sulfate in quail egg white and characteristic distribution of sulfated sugar nucleotides in different avian eggs.

    PubMed

    Nakanishi, Y; Okuda, S; Tsuji, M; Suzuki, S

    1979-08-29

    A sulfated sugar nucleotide has been isolated from quail egg white, and accounts for nearly 80% of the total sugar nucleotides found in the egg white. Evidence is presented that this nucleotide is uridine diphosphate N-acetylgalactosamine 6-sulfate, an isomer of the 4-sulfated derivative of uridine diphosphate N-acetylgalactosamine previously found in chicken egg white. Further studies on the distribution of sulfated sugar nucleotides in egg white of various birds (chicken, quail, pheasant, peafowl, turkey, goose, and duck) demonstrate that each species has a characteristic composition, differing from one another regarding the relative amounts of 4-sulfated, 6-sulfated, and 4,6-bissulfated derivatives of uridine diphosphate N-acetylgalactosamine. PMID:534643

  17. Effect of coadministration of ketoconazole, a strong CYP3A4 inhibitor, on pharmacokinetics and tolerability of motesanib diphosphate (AMG 706) in patients with advanced solid tumors.

    PubMed

    Lorusso, Patricia; Heath, Elisabeth I; McGreivy, Jesse; Sun, Yu-Nien; Melara, Rebeca; Yan, Lucy; Malburg, Lisa; Ingram, Megan; Wiezorek, Jeffrey; Chen, Li; Pilat, Mary Jo

    2008-10-01

    Motesanib diphosphate is a novel angiogenesis inhibitor selectively targeting vascular endothelial growth factor receptors 1, 2, and 3; platelet-derived growth factor receptor and stem cell factor receptor. The purpose of this phase 1b, drug-drug interaction study was to investigate the effect of ketoconazole, a strong inhibitor of the cytochrome P450 3A4 isoenzyme, on the pharmacokinetics and tolerability of motesanib diphosphate. Fourteen patients with advanced solid tumors refractory to standard treatment were enrolled and received motesanib diphosphate 50 mg once daily from day 1 through 15. Patients were randomized to receive a single oral dose of ketoconazole 400 mg either on day 8 (Sequence 1; n = 7) or day 15 (Sequence 2; n = 7), while pharmacokinetic samples were collected. After completion of this part (day 16), 13 patients received an escalated once-daily dose of motesanib diphosphate 125 mg. Evaluable pharmacokinetic data (n = 12) suggest that ketoconazole modestly increased motesanib exposure. The motesanib area under the concentration-time curve (AUC) from 0 to 24 h increased by 86% (90% CI, 1.50-2.29; P < 0.001) and the maximum plasma concentration (C (max)) by 35% (90% CI, 1.12-1.64; P = 0.02), compared with motesanib diphosphate administration alone. The tolerability profile (with or without ketoconazole coadministration) was consistent with that from other motesanib diphosphate monotherapy studies. Treatment-related adverse events were mild to moderate and commonly included fatigue (50% of patients), hypertension (43%), diarrhea (21%), dizziness (14%), paresthesia (14%), and vomiting (14%). Hypertension was the most common related grade 3 event (21%). No grade 4 or 5 treatment-related adverse events occurred. PMID:18574557

  18. Adenosine influences myeloid cells to inhibit aeroallergen sensitization.

    PubMed

    Pei, Hong; Linden, Joel

    2016-05-15

    Agonists of adenosine A2A receptors (A2ARs) suppress the activation of most immune cells and reduce acute inflammatory responses. Asthma is characterized by sensitization in response to initial allergen exposure and by airway hyperreactivity in response to allergen rechallenge. We sought to determine if A2AR activation with CGS-21680 (CGS) is more effective when CGS is administered during sensitization or rechallenge. C57BL/6 wild-type mice and Adora2a(f/f)LysMCre(+/-) mice, which lack A2ARs on myeloid cells, were sensitized with intranasal ovalbumin (OVA) and LPS. Airway sensitization was characterized by a rapid increase in numbers of IL-6(+) and IL-12(+) macrophages and dendritic cells in lungs. A2AR activation with CGS (0.1 μg·kg(-1)·min(-1) sc) only during sensitization reduced numbers of IL-6(+) and IL-12(+) myeloid cells in the lungs and reversed the effects of OVA rechallenge to increase airway hyperresponsiveness to methacholine. CGS treatment during sensitization also reduced the expansion of lung T helper (Th1 and Th17) cells and increased expansion of regulatory T cells in response to OVA rechallenge. Most of the effects of CGS administered during sensitization were eliminated by myeloid-selective A2AR deletion. Administration of CGS only during OVA rechallenge failed to reduce airway hyperresponsiveness. We conclude that myeloid cells are key targets of adenosine during sensitization and indirectly modify T cell polarization. The results suggest that a clinically useful strategy might be to use A2AR agonists to inhibit sensitization to new aeroallergens. We speculate that adenosine production by macrophages engulfing bacteria contributes to the curious suppression of sensitization in response to early-life infections. PMID:27016586

  19. Inhibition of adenosine kinase by phosphonate and bisphosphonate derivatives.

    PubMed

    Park, Jae; Singh, Bhag; Gupta, Radhey S

    2006-02-01

    The enzyme adenosine kinase (AK) plays a central role in regulating the intracellular and interstitial concentration of the purine nucleoside adenosine (Ado). In view of the beneficial effects of Ado in protecting tissues from ischemia and other stresses, there is much interest in developing AK inhibitors, which can regulate Ado concentration in a site- and event-specific manner. The catalytic activity of AK from different sources is dependent upon the presence of activators such as phosphate (Pi). In this work we describe several new phosphorylated compounds which either activate or inhibit AK. The compounds acetyl phosphate, carbamoyl phosphate, dihydroxyacetone phosphate and imidodiphosphate were found to stimulate AK activity in a dose-dependent manner comparable to that seen with Pi. In contrast, a number of phosphonate and bisphosphonate derivatives, which included clodronate and etidronate, were found to inhibit the activity of purified AK in the presence of Pi. These AK inhibitors (viz. clodronate, etidronate, phosphonoacetic acid, 2-carboxyethylphosphonic acid, N-(phosphonomethyl)-glycine and N-(phosphonomethyl)iminodiacetic acid), at concentrations at which they inhibited AK, were also shown to inhibit the uptake of (3)H-adenosine and its incorporation into macromolecules in cultured mammalian cells, indicating that they were also inhibiting AK in intact cells. The drug concentrations at which these effects were observed showed limited toxicity to the cultured cells, indicating that these effects are not caused by cellular toxicity. These results indicate that the enzyme AK provides an additional cellular target for the clinically widely used bisphosphonates and related compounds, which could possibly be exploited for a new therapeutic application. Our structure-activity studies on different AK activators and inhibitors also indicate that all of the AK activating compounds have a higher partial positive charge (delta(+)) on the central phosphorous atom in

  20. Outcomes and costs of positron emission tomography: comparison of intravenous adenosine and intravenous dipyridamole.

    PubMed

    Holmberg, M J; Mohiuddin, S M; Hilleman, D E; Lucas, B D; Wadibia, E C

    1997-01-01

    The objective of this study was to compare the cost of intravenous adenosine and intravenous dipyridamole in positron emission tomography (PET) in patients with coronary artery disease. A retrospective, open-label, case-control, cost-effectiveness analysis was performed in the out-patient nuclear medicine department of a university hospital. Thirty-six patients underwent dipyridamole PET, and 72 matched patients underwent adenosine PET. A cost-effectiveness analysis was conducted using a direct cost accounting approach to estimate institutional costs. Key costs evaluated included acquisition cost, administration cost, monitoring cost, cost of management of side effects, and cost of follow-up care. The total cost of adenosine PET and dipyridamole PET was divided by their respective predictive accuracies to provide a total cost adjusted for efficacy. Adenosine increased heart rate and lowered systolic blood pressure to a significantly greater extent than dipyridamole. The number of patients experiencing adverse drug reactions was significantly greater for adenosine (82%) than for dipyridamole (67%), but the frequency of prolonged (> 5 minutes) and late-onset side effects was significantly greater for dipyridamole than for adenosine. The frequency of side effects requiring medical intervention was also significantly greater for dipyridamole (53%) than for adenosine (6%). Although adenosine had a significantly greater acquisition cost than dipyridamole, costs of monitoring, management of side effects, and follow-up care were significantly less for adenosine than for dipyridamole. As a result, the total cost of using dipyridamole is significantly greater ($928.00 per patient) than the total cost of using adenosine ($672.00 per patient). Based on these results, adenosine may be the drug of choice for pharmacologic vasodilation for PET. PMID:9220220

  1. Presynaptic action of adenosine on a 4-aminopyridine-sensitive current in the rat carotid body

    PubMed Central

    Vandier, C; Conway, A F; Landauer, R C; Kumar, P

    1999-01-01

    Plasma adenosine concentration increases during hypoxia to a level that excites carotid body chemoreceptors by an undetermined mechanism. We have examined this further by determining the electrophysiological responses to exogenous adenosine of sinus nerve chemoafferents in vitro and of whole-cell currents in isolated type I cells.Steady-state, single-fibre chemoafferent discharge was increased approximately 5-fold above basal levels by 100 μM adenosine. This adenosine-stimulated discharge was reversibly and increasingly reduced by methoxyverapamil (D600, 100 μM), by application of nickel chloride (Ni2+, 2 mM) and by removal of extracellular Ca2+. These effects strongly suggest a presynaptic, excitatory action of adenosine on type I cells of the carotid body.Adenosine decreased whole-cell outward currents at membrane potentials above -40 mV in isolated type I cells recorded during superfusion with bicarbonate-buffered saline solution at 34–36 °C. This effect was reversible and concentration dependent with a maximal effect at 10 μM.The degree of current inhibition induced by 10 μM adenosine was voltage independent (45.39 ± 2.55% (mean ± s.e.m.) between −40 and +30 mV) and largely (∼75%), but not entirely, Ca2+ independent. 4-Aminopyridine (4-AP, 5 mM) decreased the amplitude of the control outward current by 80.60 ± 3.67% and abolished the effect of adenosine.Adenosine was without effect upon currents near the resting membrane potential of approximately −55 mV and did not induce depolarization in current-clamp experiments.We conclude that adenosine acts to inhibit a 4-AP-sensitive current in isolated type I cells of the rat carotid body and suggest that this mechanism contributes to the chemoexcitatory effect of adenosine in the whole carotid body. PMID:10050009

  2. Suppression of CYP2B Induction by Alendronate-Mediated Farnesyl Diphosphate Synthase Inhibition in Primary Cultured Rat Hepatocytes

    PubMed Central

    Jackson, Nancy M.; Kocarek, Thomas A.

    2008-01-01

    We previously reported that squalestatin 1-mediated induction of CYP2B expression is attributable to squalene synthase inhibition and accumulation of an endogenous isoprenoid(s) that is capable of activating the constitutive androstane receptor. To determine whether squalestatin 1-mediated CYP2B induction is strictly dependent upon the biosynthesis of farnesyl pyrophosphate (FPP), the substrate for squalene synthase, the effects of alendronate, a nitrogen-containing bisphosphonate inhibitor of farnesyl diphosphate synthase, were determined on basal, squalestatin 1-inducible, and phenobarbital-inducible CYP2B expression in primary cultured rat hepatocytes. Alendronate treatment alone had no effect on CYP2B or CYP3A mRNA expression in the hepatocyte cultures, but alendronate co-treatment completely suppressed squalestatin 1-mediated CYP2B mRNA induction at concentrations (60 and 100 μM) that effectively inhibited cellular farnesyl diphosphate synthase activity, as assessed by reductions of squalestatin 1-mediated FPP accumulation, and that were not toxic to the cells, as indicated by a lack of effect on MTT activity. Alendronate co-treatment also partially suppressed phenobarbital-inducible CYP2B expression, and this suppressive effect was attenuated by additional co-treatment with the upstream pathway inhibitor, pravastatin. These findings demonstrate that squalestatin 1-mediated CYP2B induction cannot occur in the absence of FPP biosynthesis, but also indicate that one or more upstream isoprenoids, possibly isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate, function to antagonize the CYP2B induction process. PMID:18617600

  3. The Thiamine diphosphate dependent Enzyme Engineering Database: A tool for the systematic analysis of sequence and structure relations

    PubMed Central

    2010-01-01

    Background Thiamine diphosphate (ThDP)-dependent enzymes form a vast and diverse class of proteins, catalyzing a wide variety of enzymatic reactions including the formation or cleavage of carbon-sulfur, carbon-oxygen, carbon-nitrogen, and especially carbon-carbon bonds. Although very diverse in sequence and domain organisation, they share two common protein domains, the pyrophosphate (PP) and the pyrimidine (PYR) domain. For the comprehensive and systematic comparison of protein sequences and structures the Thiamine diphosphate (ThDP)-dependent Enzyme Engineering Database (TEED) was established. Description The TEED http://www.teed.uni-stuttgart.de contains 12048 sequence entries which were assigned to 9443 different proteins and 379 structure entries. Proteins were assigned to 8 different superfamilies and 63 homologous protein families. For each family, the TEED offers multisequence alignments, phylogenetic trees, and family-specific HMM profiles. The conserved pyrophosphate (PP) and pyrimidine (PYR) domains have been annotated, which allows the analysis of sequence similarities for a broad variety of proteins. Human ThDP-dependent enzymes are known to be involved in many diseases. 20 different proteins and over 40 single nucleotide polymorphisms (SNPs) of human ThDP-dependent enzymes were identified in the TEED. Conclusions The online accessible version of the TEED has been designed to serve as a navigation and analysis tool for the large and diverse family of ThDP-dependent enzymes. PMID:20122171

  4. The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase.

    PubMed

    Harris, L J; Saparno, A; Johnston, A; Prisic, S; Xu, M; Allard, S; Kathiresan, A; Ouellet, T; Peters, R J

    2005-12-01

    Using the technique of differential display, a maize transcript was identified whose silk tissue expression is induced in the presence of the ear rot pathogen Fusarium graminearum. The 3445 nt transcript includes a 727 nt 5' untranslated leader with the potential for extensive secondary structure and represents the maize gene An2. An2 encodes a copalyl diphosphate synthase (CPS)-like protein with 60% amino acid sequence identity with the maize An1 gene product involved in gibberellin (GA) biosynthesis. Recombinant expression and functional analysis demonstrated that both AN1 and AN2 are ent-copalyl diphosphate (ent-CPP) synthases (ent-CPS). Notably, the presence of an additional ent-CPS gene is consistent with previous reports that maize GA biosynthesis can proceed in the absence of An1. In addition, northern blot analysis showed that An2 transcript levels were strongly up-regulated by Fusarium attack, with an increase in silk, husk and ear tip tissues as early as 6 h after inoculation of silk channels with spore suspensions of various Fusarium sp. Gene expression of a third maize CPS-like gene, Cpsl1, is not affected by Fusarium infection. The Fusarium-inducible nature of An2 is also consistent with a previous report that cell-free extracts from maize seedlings produce ent-CPP derived diterpenes in response to Fusarium infection. However, it is not known whether An2 is involved in defense-related secondary metabolism in addition to GA synthesis. PMID:16307364

  5. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    SciTech Connect

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

  6. A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

    PubMed Central

    Motomura, Kei; Hirota, Ryuichi; Okada, Mai; Ikeda, Takeshi; Ishida, Takenori

    2014-01-01

    Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by “high-energy” phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities. PMID:24532069

  7. Adenosine conjugated lipidic nanoparticles for enhanced tumor targeting.

    PubMed

    Swami, Rajan; Singh, Indu; Jeengar, Manish Kumar; Naidu, V G M; Khan, Wahid; Sistla, Ramakrishna

    2015-01-01

    Delivering chemotherapeutics by nanoparticles into tumor is impeded majorly by two factors: nonspecific targeting and inefficient penetration. Targeted delivery of anti-cancer agents solely to tumor cells introduces a smart strategy because it enhances the therapeutic index compared with untargeted drugs. The present study was performed to investigate the efficiency of adenosine (ADN) to target solid lipid nanoparticles (SLN) to over expressing adenosine receptor cell lines such as human breast cancer and prostate cancer (MCF-7 and DU-145 cells), respectively. SLN were prepared by emulsification and solvent evaporation process using docetaxel (DTX) as drug and were characterized by various techniques like dynamic light scattering, differential scanning calorimeter and transmission electron microscopy. DTX loaded SLNs were surface modified with ADN, an adenosine receptors ligand using carbodiimide coupling. Conjugation was confirmed using infrared spectroscopy and quantified using phenol-sulfuric acid method. Conjugated SLN were shown to have sustained drug release as compared to unconjugated nanoparticles and drug suspension. Compared with free DTX and unconjugated SLN, ADN conjugated SLN showed significantly higher cytotoxicity of loaded DTX, as evidenced by in vitro cell experiments. The IC50 was 0.41 μg/ml for native DTX, 0.30 μg/ml for unconjugated SLN formulation, and 0.09 μg/ml for ADN conjugated SLN formulation in MCF-7 cell lines. Whereas, in DU-145, there was 2 fold change in IC50 of ADN-SLN as compared to DTX. IC50 was found to be 0.44 μg/ml for free DTX, 0.39 μg/ml for unconjugated SLN and 0.22 μg/ml for ADN-SLN. Annexin assay and cell cycle analysis assay further substantiated the cell cytotoxicity. Fluorescent cell uptake and competitive ligand-receptor binding assay corroborated the receptor mediated endocytosis pathway indicated role of adenosine receptors in internalization of conjugated particles. Pharmacokinetic studies of lipidic

  8. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

    PubMed Central

    2014-01-01

    Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages. PMID:25105011

  9. 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.

  10. Silver vanadium diphosphate Ag{sub 2}VP{sub 2}O{sub 8}: Electrochemistry and characterization of reduced material providing mechanistic insights

    SciTech Connect

    Takeuchi, Esther S.; Lee, Chia-Ying; Cheng, Po-Jen; Menard, Melissa C.; Marschilok, Amy C.; Takeuchi, Kenneth J.

    2013-04-15

    Silver vanadium phosphorous oxides (Ag{sub w}V{sub x}P{sub y}O{sub z}) are notable battery cathode materials due to their high energy density and demonstrated ability to form in-situ Ag metal nanostructured electrically conductive networks within the cathode. While analogous silver vanadium diphosphate materials have been prepared, electrochemical evaluations of these diphosphate based materials have been limited. We report here the first electrochemical study of a silver vanadium diphosphate, Ag{sub 2}VP{sub 2}O{sub 8}, where the structural differences associated with phosphorous oxides versus diphosphates profoundly affect the associated electrochemistry. Reminiscent of Ag{sub 2}VO{sub 2}PO{sub 4} reduction, in-situ formation of silver metal nanoparticles was observed with reduction of Ag{sub 2}VP{sub 2}O{sub 8}. However, counter to Ag{sub 2}VO{sub 2}PO{sub 4} reduction, Ag{sub 2}VP{sub 2}O{sub 8} demonstrates a significant decrease in conductivity upon continued electrochemical reduction. Structural analysis contrasting the crystallography of the parent Ag{sub 2}VP{sub 2}O{sub 8} with that of the proposed Li{sub 2}VP{sub 2}O{sub 8} reduction product is employed to gain insight into the observed electrochemical reduction behavior, where the structural rigidity associated with the diphosphate anion may be associated with the observed particle fracturing upon deep electrochemical reduction. Further, the diphosphate anion structure may be associated with the high thermal stability of the partially reduced Ag{sub 2}VP{sub 2}O{sub 8} materials, which bodes well for enhanced safety of batteries incorporating this material. - Graphical abstract: Structure and galvanostatic intermittent titration-type test data for silver vanadium diphosphate, Ag{sub 2}VP{sub 2}O{sub 8}. Highlights: ► First electrochemical study of a silver vanadium diphosphate, Ag{sub 2}VP{sub 2}O{sub 8}. ► In-situ formation of Ag{sup 0} nanoparticles was observed upon electrochemical reduction.

  11. Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes

    PubMed Central

    Bolcato, Chiara; Cusan, Claudia; Pastorin, Giorgia; Cacciari, Barbara; Klotz, Karl Norbert; Morizzo, Erika

    2007-01-01

    In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach. PMID:18368532

  12. Photomodulation of G Protein-Coupled Adenosine Receptors by a Novel Light-Switchable Ligand

    PubMed Central

    2015-01-01

    The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities. PMID:25248077

  13. Spreading depolarization-induced adenosine accumulation reflects metabolic status in vitro and in vivo

    PubMed Central

    Lindquist, Britta E; Shuttleworth, C William

    2014-01-01

    Spreading depolarization (SD), a pathologic feature of migraine, stroke and traumatic brain injury, is a propagating depolarization of neurons and glia causing profound metabolic demand. Adenosine, the low-energy metabolite of ATP, has been shown to be elevated after SD in brain slices and under conditions likely to trigger SD in vivo. The relationship between metabolic status and adenosine accumulation after SD was tested here, in brain slices and in vivo. In brain slices, metabolic impairment (assessed by nicotinamide adenine dinucleotide (phosphate) autofluorescence and O2 availability) was associated with prolonged extracellular direct current (DC) shifts indicating delayed repolarization, and increased adenosine accumulation. In vivo, adenosine accumulation was observed after SD even in otherwise healthy mice. As in brain slices, in vivo adenosine accumulation correlated with DC shift duration and increased when DC shifts were prolonged by metabolic impairment (i.e., hypoglycemia or middle cerebral artery occlusion). A striking pattern of adenosine dynamics was observed during focal ischemic stroke, with nearly all the observed adenosine signals in the periinfarct region occurring in association with SDs. These findings suggest that adenosine accumulation could serve as a biomarker of SD incidence and severity, in a range of clinical conditions. PMID:25160669

  14. Adenosine inhibition of gamma-aminobutyric acid release from slices of rat cerebral cortex.

    PubMed Central

    Hollins, C.; Stone, T. W.

    1980-01-01

    1 The effect of purine compounds on the potassium-evoked release of 14C-labelled gamma-aminobutyric acid (GABA) has been studied in 400 micrometers slices of rat cerebral cortex in vitro. 2 Adenosine and adenosine 5' monophosphate (AMP) inhibited the release of GABA at 10(-5) to 10(-3) M. Adenosine triphosphate (ATP) produced a significant inhibition of release only at 10(-3) M. 3 Theophylline 10(-4) or 10(-3) M reduced the inhibitory effect of adenosine, but did not change basal release of GABA. 4 Dipyridamole 10(-5) M itself reduced evoked GABA release, but did not prevent the inhibitory effect of adenosine, implying that adenosine was acting at an extracellularly directed receptor. 5 Calcium removal or antagonism by verapamil reduced the evoked release of GABA, but adenosine did not produce any further reduction of the calcium-independent release. This may indicate that the inhibitory effect of adenosine on GABA release results from interference with calcium influx or availability within the terminals. PMID:7378648

  15. Adenosine-Activated Nanochannels Inspired by G-Protein-Coupled Receptors.

    PubMed

    Li, Pei; Kong, Xiang-Yu; Xie, Ganhua; Xiao, Kai; Zhang, Zhen; Wen, Liping; Jiang, Lei

    2016-04-01

    A bioinspired adenosine activated nanodevice is demonstrated in which the conformations of the designed aptamer change and cause signal transmission according to the emergence of adenosine. This bioinspired system exhibits very high response ratios (activated/nonactivated ratio up to 614) and excellent stability and reversibility, and shows promising applications in the fields of biosensors, pharmaceutica, and healthcare systems. PMID:26915491

  16. Inhibition of renal Na+, K+-adenosine triphosphatase by gentamicin

    SciTech Connect

    Williams, P.D.; Trimble, M.E.; Crespo, L.; Holohan, P.D.; Freedman, J.C.; Ross, C.R.

    1984-11-01

    Inhibition of renal Na+,K+-adenosine triphosphatase is an early biochemical manifestation of gentamicin treatment in rats. Studies with isolated, perfused rat kidneys in filtering and nonfiltering modes indicate that gentamicin is transported across the brush border membrane before enzyme inhibition. The drug caused enzyme inhibition (42%) only in filtering kidneys, and this inhibition was blocked by spermine, an inhibitor of gentamicin binding. In purified rat renal basolateral membranes, bound (/sup 3/H)gentamicin was displaced 88% by unlabeled gentamicin. After in vivo exposure to (/sup 3/H)gentamicin, the radioactivity associated with the isolated basolateral membranes was displaced only 46% by unlabeled drug. These results suggest that inhibition of renal Na+,K+-adenosine triphosphatase by gentamicin is probably due to an interaction at the cytoplasmic face of the basolateral membrane. Scatchard plots of (/sup 3/H)gentamicin binding to basolateral and brush border membranes revealed a single class of noninteracting sites in each membrane. Gentamicin did not change the bulk membrane lipid fluidity, as estimated by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene.

  17. Adenosine signaling and the energetic costs of induced immunity.

    PubMed

    Lazzaro, Brian P

    2015-04-01

    Life history theory predicts that trait evolution should be constrained by competing physiological demands on an organism. Immune defense provides a classic example in which immune responses are presumed to be costly and therefore come at the expense of other traits related to fitness. One strategy for mitigating the costs of expensive traits is to render them inducible, such that the cost is paid only when the trait is utilized. In the current issue of PLOS Biology, Bajgar and colleagues elegantly demonstrate the energetic and life history cost of the immune response that Drosophila melanogaster larvae induce after infection by the parasitoid wasp Leptopilina boulardi. These authors show that infection-induced proliferation of defensive blood cells commands a diversion of dietary carbon away from somatic growth and development, with simple sugars instead being shunted to the hematopoetic organ for rapid conversion into the raw energy required for cell proliferation. This metabolic shift results in a 15% delay in the development of the infected larva and is mediated by adenosine signaling between the hematopoietic organ and the central metabolic control organ of the host fly. The adenosine signal thus allows D. melanogaster to rapidly marshal the energy needed for effective defense and to pay the cost of immunity only when infected. PMID:25915419

  18. Adenosine Amine Congener as a Cochlear Rescue Agent

    PubMed Central

    Vlajkovic, Srdjan M.; Chang, Hao; Paek, Song Yee; Chi, Howard H.-T.; Sreebhavan, Sreevalsan; Telang, Ravindra S.; Tingle, Malcolm; Housley, Gary D.; Thorne, Peter R.

    2014-01-01

    We have previously shown that adenosine amine congener (ADAC), a selective A1 adenosine receptor agonist, can ameliorate noise- and cisplatin-induced cochlear injury. Here we demonstrate the dose-dependent rescue effects of ADAC on noise-induced cochlear injury in a rat model and establish the time window for treatment. Methods. ADAC (25–300 μg/kg) was administered intraperitoneally to Wistar rats (8–10 weeks old) at intervals (6–72 hours) after exposure to traumatic noise (8–16 kHz, 110 dB sound pressure level, 2 hours). Hearing sensitivity was assessed using auditory brainstem responses (ABR) before and 12 days after noise exposure. Pharmacokinetic studies investigated ADAC concentrations in plasma after systemic (intravenous) administration. Results. ADAC was most effective in the first 24 hours after noise exposure at doses >50 μg/kg, providing up to 21 dB protection (averaged across 8–28 kHz). Pharmacokinetic studies demonstrated a short (5 min) half-life of ADAC in plasma after intravenous administration without detection of degradation products. Conclusion. Our data show that ADAC mitigates noise-induced hearing loss in a dose- and time-dependent manner, but further studies are required to establish its translation as a clinical otological treatment. PMID:25243188

  19. Purification and characterization of Plasmodium yoelii adenosine deaminase.

    PubMed

    Yadav, Sarika; Saxena, Jitendra Kumar; Dwivedi, U N

    2011-12-01

    Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K(m) values of 41 μM and 34 μM for adenosine and 2'-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K(i) value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional -SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA. PMID:21945268

  20. Adenosine-to-inosine RNA editing and human disease

    PubMed Central

    2013-01-01

    A-to-I RNA editing is a post-transcriptional modification that converts adenosines to inosines in both coding and noncoding RNA transcripts. It is catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes, which exist throughout the body but are most prevalent in the central nervous system. Inosines exhibit properties that are most similar to those of guanosines. As a result, ADAR-mediated editing can post-transcriptionally alter codons, introduce or remove splice sites, or affect the base pairing of the RNA molecule with itself or with other RNAs. A-to-I editing is a mechanism that regulates and diversifies the transcriptome, but the full biological significance of ADARs is not understood. ADARs are highly conserved across vertebrates and are essential for normal development in mammals. Aberrant ADAR activity has been associated with a wide range of human diseases, including cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders. ADARs have been shown to contribute to disease pathologies by editing of glutamate receptors, editing of serotonin receptors, mutations in ADAR genes, and by other mechanisms, including recently identified regulatory roles in microRNA processing. Advances in research into many of these diseases may depend on an improved understanding of the biological functions of ADARs. Here, we review recent studies investigating connections between ADAR-mediated RNA editing and human diseases. PMID:24289319

  1. Distribution of adenosine receptors in human sclera fibroblasts

    PubMed Central

    Cui, Dongmei; Trier, Klaus; Chen, Xiang; Zeng, Junwen; Yang, Xiao; Hu, Jianmin

    2008-01-01

    Purpose Systemic treatment with adenosine receptor antagonists has been reported to affect the biochemistry and ultrastructure of rabbit sclera. This study was conducted to determine whether adenosine receptors (ADORs) are present in human scleral fibroblasts (HSF). Methods Primary HSF were cultured in vitro and identified with anti-vimentin, anti-keratin, anti-desmin, and anti-S-100 antibodies. Confocal fluorescence microscopy was used to study the distribution of ADORs in the HSF cell lines and in the frozen human scleral sections. ADOR protein expression in HSF and human sclera was confirmed by western blot analysis of cell lysates. Results ADORs were expressed in both HSF and human sclera. This was confirmed by western blot. ADORA1 expression was concentrated in the nucleus. ADORA2A was concentrated mainly in one side of the cytoplasm, and ADORA2B was found both in the nucleus and the cytoplasm. ADORA3 was expressed weakly in the cytoplasm. Conclusions All four subtypes of ADOR were found in HSF and may play a role in scleral remodeling. PMID:18385786

  2. Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

    PubMed

    Hill, R J; Oleynek, J J; Hoth, C F; Kiron, M A; Weng, W; Wester, R T; Tracey, W R; Knight, D R; Buchholz, R A; Kennedy, S P

    1997-01-01

    The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in

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

    PubMed

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

    2016-07-19

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

  4. Novel anti-thrombotic agent for modulation of protein disulfide isomerase family member ERp57 for prophylactic therapy

    PubMed Central

    Cui, Guozhen; Shan, Luchen; Guo, Lin; Chu, Ivan Keung; Li, Guohui; Quan, Quan; Zhao, Yun; Chong, Cheong Meng; Zhang, Zaijun; Yu, Pei; Hoi, Maggie Pui Man; Sun, Yewei; Wang, Yuqiang; Lee, Simon MingYuen

    2015-01-01

    Protein disulfide isomerase (PDI) family members including PDI and ERp57 emerge as novel targets for anti-thrombotic treatments, but chemical agents with selectivity remain to be explored. We previously reported a novel derivative of danshensu (DSS), known as ADTM, displayed strong cardioprotective effects against oxidative stress-induced cellular injury in vitro and acute myocardial infarct in vivo. Herein, using chemical proteomics approach, we identified ERp57 as a major target of ADTM. ADTM displayed potent inhibitory effects on the redox activity of ERp57, inhibited the adenosine diphosphate (ADP)-induced expressions of P-selectin and αIIbβ3 integrin, and disrupted the interaction between ERp57 and αIIbβ3. In addition, ADTM inhibited both arachidonic acid (AA)-induced and ADP-induced platelet aggregation in vitro. Furthermore, ADTM significantly inhibited rat platelet aggregation and thrombus formation in vivo. Taken together, ADTM represents a promising candidate for anti-thrombotic therapy targeting ERp57. PMID:26037049

  5. Production and characterization of monoclonal antibodies against rat platelet GPIIb/IIIa

    SciTech Connect

    Miyazaki, H.; Tamura, S.; Sudo, T.; Suzuki, T. )

    1990-09-15

    Four murine monoclonal antibodies against rat platelets were produced by fusion of spleen cells from mice intravenously immunized with whole rat platelets. All four antibodies immunoprecipitated two major platelet membrane proteins with apparent molecular weights of 130,000 and 82,000 (nonreduced) and of 120,000 and 98,000 (reduced), which were structurally analogous to human glycoprotein (GP) IIb/IIIa, i.e. rat GPIIb/IIIa. Two of four antibodies, named P9 and P55, strongly inhibited adenosine diphosphate (ADP)-induced aggregation of washed rat platelets and caused approximately 50% inhibition of human fibrinogen binding to ADP-stimulated rat platelets, suggesting that rat GPIIb/IIIa serves as a fibrinogen receptor in ADP-induced aggregation. In contrast, two other antibodies, named P14 and P34, themselves caused aggregation of rat platelets in platelet-rich plasma (PRP) and the secretion of 14C-serotonin from 14C-serotonin-labeled PRP. These results indicate that rat GPIIb/IIIa plays an important role in platelet aggregation.

  6. Purification and Properties of Adenosine Diphosphoglucose Pyrophosphorylase from Sweet Corn 1

    PubMed Central

    Amir, Jacob; Cherry, Joe H.

    1972-01-01

    A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg2+ and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1-phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 × 10−4, 3.2 × 10−5, 3.3 × 10−5, and 6.2 × 10−4m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 × 10−7m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate. PMID:16658078

  7. Respiratory stimulant effects of adenosine in man after caffeine and enprofylline.

    PubMed Central

    Smits, P; Schouten, J; Thien, T

    1987-01-01

    In a double-blind and randomized study the respiratory stimulant effect of continuous intravenous adenosine infusion was studied after previous administration of caffeine, placebo and enprofylline in 10 healthy young volunteers. After placebo, adenosine induced an increase of minute ventilation (from 6.3 to 12.5 l min-1), tidal volume (from 0.60 to 0.96 l), and breathing rate (from 11.0 to 14.8 min-1). Venous pCO2 fell and pH rose after adenosine. Caffeine significantly reduced the adenosine-induced changes of minute ventilation, tidal volume, venous pCO2 and pH, whereas no changes occurred after enprofylline. Our results suggest that adenosine stimulates respiration in man by binding with specific P1-purinoceptors, which can be blocked by caffeine, but not by enprofylline. PMID:3440102

  8. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Ng, Seng Kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2015-05-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR(+/-)) significantly protect ESMN from SOD1G93A(+) astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS. PMID:25779930

  9. 2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

    PubMed

    Konno, Takashi; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-08-22

    The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production. PMID:16023100

  10. Involvement of adenosine A2a receptor in intraocular pressure decrease induced by 2-(1-octyn-1-yl)adenosine or 2-(6-cyano-1-hexyn-1-yl)adenosine.

    PubMed

    Konno, Takashi; Murakami, Akira; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-04-01

    The aim of the present study is to clarify the mechanism for the decrease in intraocular pressure by 2-alkynyladenosine derivatives in rabbits. The receptor binding analysis revealed that 2-(1-octyn-1-yl)adenosine (2-O-Ado) and 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) selectively bound to the A(2a) receptor with a high affinity. Ocular hypotensive responses to 2-O-Ado and 2-CN-Ado were inhibited by the adenosine A(2a)-receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), but not by the adenosine A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the adenosine A(2b)-receptor antagonist alloxazine. In addition, 2-O-Ado and 2-CN-Ado caused an increase in outflow facility, which was inhibited by CSC, but not by DPCPX or alloxazine. Moreover, 2-O-Ado and 2-CN-Ado increased cAMP in the aqueous humor, and the 2-O-Ado-induced an increase in cAMP was inhibited by CSC. These results suggest that 2-O-Ado and 2-CN-Ado reduced intraocular pressure via an increase in outflow facility. The ocular hypotension may be mainly mediated through the activation of adenosine A(2a) receptor, although a possible involvement of adenosine A(1) receptor cannot be completely ruled out. 2-O-Ado and 2-CN-Ado are useful lead compounds for the treatment of glaucoma. PMID:15821340

  11. Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

    PubMed

    Gaudin, Alice; Lepetre-Mouelhi, Sinda; Mougin, Julie; Parrod, Martine; Pieters, Grégory; Garcia-Argote, Sébastien; Loreau, Olivier; Goncalves, Jordan; Chacun, Hélène; Courbebaisse, Yann; Clayette, Pascal; Desmaële, Didier; Rousseau, Bernard; Andrieux, Karine; Couvreur, Patrick

    2015-08-28

    Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury. PMID:26087468

  12. Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis

    PubMed Central

    Gaudin, Alice; Lepetre-Mouelhi, Sinda; Mougin, Julie; Parrod, Martine; Pieters, Grégory; Garcia-Argote, Sébastien; Loreau, Olivier; Goncalves, Jordan; Chacun, Hélène; Courbebaisse, Yann; Clayette, Pascal; Desmaële, Didier; Rousseau, Bernard; Andrieux, Karine; Couvreur, Patrick

    2015-01-01

    Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[3H]-Adenosine NAs and [14C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1 h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury. PMID:26087468

  13. Specificity of synergistic coronary flow enhancement by adenosine and pulsatile perfusion in the dog.

    PubMed

    Pagliaro, P; Senzaki, H; Paolocci, N; Isoda, T; Sunagawa, G; Recchia, F A; Kass, D A

    1999-10-01

    1. Coronary flow elevation from enhanced perfusion pulsatility is synergistically amplified by adenosine. This study determined the specificity of this interaction and its potential mechanisms. 2. Mean and phasic coronary flow responses to increasing pulsatile perfusion were assessed in anaesthetized dogs, with the anterior descending coronary artery servoperfused to regulate real-time physiological flow pulsatility at constant mean pressure. Pulsatility was varied between 40 and 100 mmHg. Hearts ejected into the native aorta whilst maintaining stable loading. 3. Increasing pulsatility elevated mean coronary flow +11.5 +/- 1.7 % under basal conditions. Co-infusion of adenosine sufficient to raise baseline flow 66 % markedly amplified this pulsatile perfusion response (+82. 6 +/- 14.3 % increase in mean flow above adenosine baseline), due to a leftward shift of the adenosine-coronary flow response curve at higher pulsatility. Flow augmentation with pulsatility was not linked to higher regional oxygen consumption, supporting direct rather than metabolically driven mechanisms. 4. Neither bradykinin, acetylcholine nor verapamil reproduced the synergistic amplification of mean flow by adenosine and higher pulsatility, despite being administered at doses matching basal flow change with adenosine. 5. ATP-sensitive potassium (KATP) activation (pinacidil) amplified the pulse-flow response 3-fold, although this remained significantly less than with adenosine. Co-administration of the phospholipase A2 inhibitor quinacrine virtually eliminated adenosine-induced vasodilatation, yet synergistic interaction between adenosine and pulse perfusion persisted, albeit at a reduced level. 6. Thus, adenosine and perfusion pulsatility specifically interact to enhance coronary flow. This synergy is partially explained by KATP agonist action and additional non-flow-dependent mechanisms, and may be important for modulating flow reserve during exercise or other high output states where

  14. Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells

    PubMed Central

    Hassanian, Seyed Mahdi; Dinarvand, Peyman; Rezaie, Alireza R.

    2014-01-01

    The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B>A2A>A1>A3) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A-specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1 and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1 and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. PMID:24477600

  15. Real-time monitoring of extracellular adenosine using enzyme-linked microelectrode arrays.

    PubMed

    Hinzman, Jason M; Gibson, Justin L; Tackla, Ryan D; Costello, Mark S; Burmeister, Jason J; Quintero, Jorge E; Gerhardt, Greg A; Hartings, Jed A

    2015-12-15

    Throughout the central nervous system extracellular adenosine serves important neuroprotective and neuromodulatory functions. However, current understanding of the in vivo regulation and effects of adenosine is limited by the spatial and temporal resolution of available measurement techniques. Here, we describe an enzyme-linked microelectrode array (MEA) with high spatial (7500 µm(2)) and temporal (4 Hz) resolution that can selectively measure extracellular adenosine through the use of self-referenced coating scheme that accounts for interfering substances and the enzymatic breakdown products of adenosine. In vitro, the MEAs selectively measured adenosine in a linear fashion (r(2)=0.98±0.01, concentration range=0-15 µM, limit of detection =0.96±0.5 µM). In vivo the limit of detection was 0.04±0.02 µM, which permitted real-time monitoring of the basal extracellular concentration in rat cerebral cortex (4.3±1.5 µM). Local cortical injection of adenosine through a micropipette produced dose-dependent transient increases in the measured extracellular concentration (200 nL: 6.8±1.8 µM; 400 nL: 19.4±5.3 µM) [P<0.001]. Lastly, local injection of dipyridamole, which inhibits transport of adenosine through equilibrative nucleoside transporter, raised the measured extracellular concentration of adenosine by 120% (5.6→12.3 µM) [P<0.001]. These studies demonstrate that MEAs can selectively measure adenosine on temporal and spatial scales relevant to adenosine signaling and regulation in normal and pathologic states. PMID:26183072

  16. Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism

    PubMed Central

    Wen, Jiaming; Jiang, Xianzhen; Dai, Yingbo; Zhang, Yujin; Tang, Yuxin; Sun, Hong; Mi, Tiejuan; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2010-01-01

    Introduction Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility. Aim The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism. Methods Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol–modified ADA (PEG–ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG–ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation. Main Outcome Measures Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays. Results We found that lowering adenosine levels in penile tissues by PEG–ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG–ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism. Conclusions Our studies have identified that PEG–ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation

  17. Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway.

    PubMed

    Rekittke, Ingo; Wiesner, Jochen; Röhrich, Rene; Demmer, Ulrike; Warkentin, Eberhard; Xu, Weiya; Troschke, Kathrin; Hintz, Martin; No, Joo Hwan; Duin, Evert C; Oldfield, Eric; Jomaa, Hassan; Ermler, Ulrich

    2008-12-24

    Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 42 and His 124) and a totally conserved Glu (Glu126) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu126 provides the protons required for IPP/DMAPP formation. PMID:19035630

  18. In vitro and in vivo characterization of a novel insect decaprenyl diphosphate synthase: a two-major step catalytic mechanism is proposed.

    PubMed

    Zhang, Hao; Li, Zheng-Xi

    2013-12-01

    Medium- and long-chain polyprenyl diphosphate synthases (PDDSs) catalyze the synthesis of the side-chain prenyl tails of ubiquinones, which play critical physiological roles in all organisms. This class of enzymes has been extensively studied in bacteria, yeast, plants and mammals, but very little information about such enzymes is available in insects. Here we cloned the cDNAs encoding the two subunits of an aphid long-chain PDDS (designated as AgDPPS1 and AgDPPS2). AgDPPS1 and AgDPPS2 had an open reading frame of 1230 bp and 1275 bp, with a calculated isoelectric point of 8.13 and 6.28, respectively. Sequence alignment and phylogenetic analysis showed that the enzyme was a candidate decaprenyl diphosphate (DPP) synthase with two heterologous subunits. Recombinant expression and in vitro enzymatic assay revealed that the two subunits were essential for the activity of the enzyme that catalyzed the formation of a major intermediate product geranylgeranyl diphosphate. In vivo analysis of ubiquinone (UQ) by expressing the insect enzyme in Escherichia coli identified UQ-10. Our data suggested that the insect enzyme is a novel DPP synthase with a two-major step catalytic mechanism, which catalyzes the formation of DPP as the final product, with geranylgeranyl diphosphate as the major intermediate product. This is the first characterization of an insect long-chain DPPS that synthesizes the side-chain of coenzyme Q-10. PMID:24246678

  19. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    PubMed

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms. PMID:26941091

  20. Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

    PubMed

    Yamamura, Y; Mizuguchi, Y; Taura, F; Kurosaki, F

    2014-10-01

    A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells. PMID:25027024

  1. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    PubMed

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. PMID:21517848

  2. [Anti-platelet actions of salicylates: in vivo, ex vivo and in vitro effects of choline salicylate].

    PubMed

    Irino, O; Saitoh, K; Ohkubo, K

    1985-07-01

    Effects of choline salicylate, sodium salicylate, choline chloride and acetylsalicylic acid on platelet aggregation in vivo, ex vivo and in vitro in mice were studied. These drugs all inhibited adenosine diphosphate (ADP)-induced respiratory depression, which is closely related to platelet aggregation in vivo, with choline salicylate showing the strongest inhibitory effect. Choline salicylate had a tendency to reduce the mortality of animals injected intravenously with endotoxin, but the other drugs had no such effect. The inhibitory effects of these drugs on ADP-induced platelet aggregation ex vivo were in the order of choline salicylate greater than acetylsalicylic acid congruent to sodium salicylate greater than choline chloride congruent to no effect, and plasma concentrations of protein-unbound salicylic acid at 1 hr after oral administration of drugs were in the order of choline salicylate greater than acetylsalicylic acid congruent to sodium salicylate. The in vitro effects of these drugs were in the order of choline salicylate congruent to sodium salicylate greater than choline chloride congruent to acetylsalicylic acid congruent to no effect. Therefore, it was considered that salicylic acid played an important role on the in vivo, ex vivo and in vitro effects of choline salicylate and that choline increased plasma concentrations of salicylic acid and consequently enhanced the in vivo and ex vivo effects of salicylic acid. Furthermore, the ex vivo effects of choline salicylate were found when ADP-induced platelet aggregation was measured with platelet-rich plasma prepared from blood collected with heparin as anti-coagulant, but not when blood was collected with citrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4043866

  3. HbIDI, SlIDI and EcIDI: A comparative study of isopentenyl diphosphate isomerase activity and structure.

    PubMed

    Berthelot, Karine; Estevez, Yannick; Quiliano, Miguel; Baldera-Aguayo, Pedro A; Zimic, Mirko; Pribat, Anne; Bakleh, Marc-Elias; Teyssier, Emeline; Gallusci, Philippe; Gardrat, Christian; Lecomte, Sophie; Peruch, Frédéric

    2016-08-01

    In this study, we cloned, expressed and purified the isopentenyl diphosphate isomerases (IDIs) from two plants, Hevea brasiliensis and Solanum lycopersicum, and compared them to the already well characterized Escherichia coli IDI. Phylogenetic analysis showed high homology between the three enzymes. Their catalytic activity was investigated in vitro with recombinant purified enzymes and in vivo by complementation colorimetric tests. The three enzymes displayed consistent activities both in vitro and in vivo. In term of structure, studied by ATR-FTIR and molecular modeling, it is clear that both plant enzymes are more related to their human homologue than to E. coli IDI. But it is assumed that EcIDI represent the minimalistic part of the catalytic core, as both plant enzymes present a supplementary sequence forming an extra α-helice surrounding the catalytic site that could facilitate the biocatalysis. New potential biotechnological applications may be envisaged. PMID:27163845

  4. Loop dynamics of thymidine diphosphate-rhamnose 3'-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis.

    PubMed

    Han, Lu; Singh, Shanteri; Thorson, Jon S; Phillips, George N

    2016-01-01

    Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3'-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3'-O-methyltransferase (CalS11) catalyses a 3'-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present the crystal structure of the apo form of CalS11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis. PMID:26958582

  5. Is capillary electrophoresis on microchip devices able to genotype uridine diphosphate glucuronosyltransferase 1A1 TATA-box polymorphisms?

    PubMed

    Minucci, Angelo; Canu, Giulia; De Bonis, Maria; Delibato, Elisabetta; Capoluongo, Ettore

    2014-06-01

    In this commentary, we focused our attention on capillary electrophoresis. It achieves the efficient separation of molecular species by the application of high voltages to samples in solution. Actually, capillary electrophoresis can be performed on microchip devices, based on an automated and miniaturized electrophoresis system, based on lab-on-a-chip technology. By this technology it is possible to separate nucleic acid fragments (DNA or RNA) with respect to sizing accuracy and sizing resolution. Currently, two automated capillary electrophoresis on microchips devices are available: the Agilent 2100 Bioanalyzer and the Experion™ Automated Electrophoresis System. In this study, we evaluated if the CE is able to distinguish the three uridine diphosphate glucuronosyltransferase 1A1 TATA-box genotypes. PMID:24687976

  6. Reaction mechanisms of thiamin diphosphate enzymes: defining states of ionization and tautomerization of the cofactor at individual steps

    PubMed Central

    Nemeria, Natalia S.; Chakraborty, Sumit; Balakrishnan, Anand; Jordan, Frank

    2009-01-01

    We summarize the currently available information regarding the state of ionization and tautomerization of the 4′-aminopyrimidine ring of the thiamine diphosphate on enzymes requiring this coenzyme. This coenzyme forms a series of covalent intermediates with its substrates as an electrophilic catalyst, and the coenzyme itself also carries out intramolecular proton transfers, which is virtually unprecedented in coenzyme chemistry. An understanding of the state of ionization and tautomerization of the 4′-aminopyrimidine ring in each of these intermediates provides important details about proton movements during catalysis. CD spectroscopy, both steady-state and time-resolved, has proved crucial for obtaining this information because no other experimental method has provided such atomic detail so far. PMID:19476485

  7. Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum.

    PubMed

    Rekittke, Ingo; Olkhova, Elena; Wiesner, Jochen; Demmer, Ulrike; Warkentin, Eberhard; Jomaa, Hassan; Ermler, Ulrich

    2013-12-11

    Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe-2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs. PMID:24188825

  8. Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from D-fructose 1,6-diphosphate.

    PubMed

    Hauck, Tobias; Landmann, Christian; Raab, Thomas; Brühlmann, Fredi; Schwab, Wilfried

    2002-07-16

    The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from D-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC-DAD and HPLC-UV-MS/MS. The temperature optimum for HDF formation was 30 degrees C, whereas the pH value (pH 3-10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and D-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-(2)H(2)]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-(13)C]-D-fructose 1,6-diphosphate with [4R,S-(2)H(2)]-NADH revealed that the hydride is transferred to C-5 or C-6 of the D-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from D-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated. PMID:12110193

  9. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    PubMed Central

    Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric; Kaiser, Marcel; Amzel, L. Mario; Gabelli, Sandra B.

    2014-01-01

    Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphos­phonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca2+ ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg2+ ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures. PMID:24598749

  10. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates.

    PubMed

    Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric; Kaiser, Marcel; Amzel, L Mario; Gabelli, Sandra B

    2014-03-01

    Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca(2+) ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg(2+) ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS-46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures. PMID:24598749

  11. Role of adenosine signalling and metabolism in β-cell regeneration

    SciTech Connect

    Andersson, Olov

    2014-02-01

    Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?.

  12. Adenosine, ketogenic diet and epilepsy: the emerging therapeutic relationship between metabolism and brain activity.

    PubMed

    Masino, S A; Kawamura, M; Wasser, C D; Wasser, C A; Pomeroy, L T; Ruskin, D N

    2009-09-01

    For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a "retaliatory metabolite." As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor-based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed. PMID:20190967

  13. The Role of Adenosine in Pulmonary Vein Isolation: A Critical Review

    PubMed Central

    Dallaglio, Paolo D.; Betts, Timothy R.; Ginks, Matthew; Bashir, Yaver; Anguera, Ignasi; Rajappan, Kim

    2016-01-01

    The cornerstone of atrial fibrillation (AF) ablation is pulmonary vein isolation (PVI), which can be achieved in more than 95% of patients at the end of the procedure. However, AF recurrence rates remain high and are related to recovery of PV conduction. Adenosine testing is used to unmask dormant pulmonary vein conduction (DC). The aim of this study is to review the available literature addressing the role of adenosine testing and determine the impact of ablation at sites of PV reconnection on freedom from AF. Adenosine infusion, by restoring the excitability threshold, unmasks reversible injury that could lead to recovery of PV conduction. The studies included in this review suggest that adenosine is useful to unmask nontransmural lesions at risk of reconnection and that further ablation at sites of DC is associated with improvement in freedom from AF. Nevertheless it has been demonstrated that adenosine is not able to predict all veins at risk of later reconnection, which means that veins without DC are not necessarily at low risk. The role of the waiting period in the setting of adenosine testing has also been analyzed, suggesting that in the acute phase adenosine use should be accompanied by enough waiting time. PMID:26981309

  14. Susceptibility to seizure-induced sudden death in DBA/2 mice is altered by adenosine.

    PubMed

    Faingold, Carl L; Randall, Marc; Kommajosyula, Srinivasa P

    2016-08-01

    Sudden unexpected death in epilepsy (SUDEP) is rare but is an important public health burden due to the number of patient years lost. Respiratory dysfunction following generalized convulsive seizure is a common sequence of events in witnessed SUDEP cases. The DBA/2 mouse model of SUDEP exhibits generalized convulsive audiogenic seizures (AGSz), which result in seizure-induced respiratory arrest (S-IRA) in ∼75% of these animals, while the remaining DBA/2 mice exhibit AGSz without S-IRA. SUDEP induction may involve actions of adenosine, which is released during generalized seizures in animals and patients and is known to depress respiration. This study examined the effects of systemic administration of agents that alter the actions of adenosine on the incidence of S-IRA in DBA/2 mice. DBA/2 mice that consistently exhibited AGSz without S-IRA showed a significantly increased incidence of S-IRA following treatment with 5-iodotubercidin, which blocks adenosine metabolism. Treatment of DBA/2 mice that consistently exhibited AGSz followed by S-IRA with a non-selective adenosine antagonist, caffeine, or an A2A adenosine receptor subtype-selective antagonist (SCH 442416) significantly reduced S-IRA incidence. By contrast, an A1 adenosine receptor antagonist (DPCPX) was not effective in reducing S-IRA incidence. These findings suggest that preventative approaches for SUDEP should consider agents that reduce the actions of adenosine. PMID:27259068

  15. Topical adenosine increases the proportion of thick hair in Caucasian men with androgenetic alopecia.

    PubMed

    Iwabuchi, Tokuro; Ideta, Ritsuro; Ehama, Ritsuko; Yamanishi, Haruyo; Iino, Masato; Nakazawa, Yosuke; Kobayashi, Takashi; Ohyama, Manabu; Kishimoto, Jiro

    2016-05-01

    Adenosine is an effective treatment for androgenetic alopecia (AGA) in Japanese men and women. Adenosine exerts its effects by significantly increasing the proportion of thick hair. In this study, we assessed the clinical outcome of adenosine treatment for 6 months in 38 Caucasian men. The change in proportion of thick hair (≥60 μm) compared with baseline in the adenosine group was significantly higher than that in the placebo group (P < 0.0001). The change in vellus hair proportion (<40 μm) was significantly lower in the adenosine group than that in the placebo group (P = 0.0154). The change in hair density compared with baseline of the adenosine group was also significantly higher compared with that of the placebo group (P = 0.0470). No adverse effects due to treatment were noted during this study by dermatological evaluation. Adenosine is effective in increasing the proportion of thick hair in Caucasian men with AGA as well as in Japanese men and women. PMID:26508659

  16. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    SciTech Connect

    Schousboe, A.; Frandsen, A.; Drejer, J. )

    1989-09-01

    Evoked release of ({sup 3}H)-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and (3H)-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.

  17. Improvement of Cold Tolerance by Selective A1 Adenosine Receptor Antagonists in Rats

    PubMed Central

    Lee, T. F.; Li, D. J.; Jacobson, K. A.; Wang, L. C. H.

    2015-01-01

    Previously we have shown that the improvement of cold tolerance by theophylline is due to antagonism at adenosine receptors rather than inhibition of phosphodiesterase. Since theophylline is a nonselective adenosine receptor antagonist for both A1 and A2 receptors, the present study investigated the adenosine receptor subtype involved in theophylline’s action. Acute systemic injection of selective A1 receptor antagonists (1,3-dialkyl-8-aryl or 1,3-dialkyl-8-cyclopentyl xanthine derivatives) significantly increased both the total and maximal heat production as well as cold tolerance. In contrast, injection of a relatively selective A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (compound No. 19), failed to significantly alter the thermogenic response of the rat under cold exposure. Further, the relative effectiveness of these compounds in increasing total thermogenesis was positively correlated with their potency in blocking the A1 adenosine receptor (r= .52, p<0.01), but not in A2 adenosine receptor (r= .20, p<0.2). It is likely that the thermally beneficial effects of adenosine A1 antagonists are due to their attenuation of the inhibitory effects of endogenously released adenosine on lipolysis and glucose utilization, resulting in increased substrate mobilization and utilization for enhanced thermogenesis. PMID:2263650

  18. Expression of adenosine A2b receptor in rat type II and III taste cells.

    PubMed

    Nishida, Kentaro; Dohi, Yukari; Yamanaka, Yuri; Miyata, Ai; Tsukamoto, Katsunobu; Yabu, Miharu; Ohishi, Akihiro; Nagasawa, Kazuki

    2014-05-01

    We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats. PMID:24327108

  19. Adenosine-5'-phosphosulfate kinase is essential for Arabidopsis viability.

    PubMed

    Mugford, Sarah G; Matthewman, Colette A; Hill, Lionel; Kopriva, Stanislav

    2010-01-01

    In Arabidopsis thaliana, adenosine-5'-phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability. PMID:19903478

  20. N6-adenosine methylation in MiRNAs.

    PubMed

    Berulava, Tea; Rahmann, Sven; Rademacher, Katrin; Klein-Hitpass, Ludgar; Horsthemke, Bernhard

    2015-01-01

    Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression. PMID:25723394

  1. N6-Adenosine Methylation in MiRNAs

    PubMed Central

    Berulava, Tea; Rahmann, Sven; Rademacher, Katrin; Klein-Hitpass, Ludgar; Horsthemke, Bernhard

    2015-01-01

    Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression. PMID:25723394

  2. Role of adenosine A2B receptors in inflammation

    PubMed Central

    Feoktistov, Igor; Biaggioni, Italo

    2013-01-01

    Recent progress in our understanding of the unique role of A2B receptors in the regulation of inflammation, immunity and tissue repair was considerably facilitated with the introduction of new pharmacological and genetic tools. However, it also led to seemingly conflicting conclusions on the role of A2B adenosine receptors in inflammation with some publications indicating pro-inflammatory effects and others suggesting the opposite. This chapter reviews the functions of A2B receptors in various cell types related to inflammation and integrated effects of A2B receptor modulation in several animal models of inflammation. It is argued that translation of current findings into novel therapies would require a better understanding of A2B receptors functions in diverse types of inflammatory responses in various tissues and at different points of their progression. PMID:21586358

  3. 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)

  4. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

    2009-01-01

    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  5. Structural and Metabolic Specificity of Methylthiocoformycin for Malarial Adenosine Deaminases

    SciTech Connect

    Ho, M.; Cassera, M; Madrid, D; Ting, L; Tyler, P; Kim, K; Almo, S; Schramm, V

    2009-01-01

    Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5?-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5?-methylthioribosyl groups are rotated 130 degrees. A hydrogen bonding network between Asp172 and the 3?-hydroxyl of MT-coformycin is essential for recognition of the 5?-methylthioribosyl group. Water occupies the 5?-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

  6. Vasodilator effects of adenosine on retinal arterioles in streptozotocin-induced diabetic rats.

    PubMed

    Nakazawa, Taisuke; Mori, Asami; Saito, Maki; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2008-02-01

    Adenosine is a potent vasodilator of retinal blood vessels and is implicated to be a major regulator of retinal blood flow during metabolic stress, but little is known about the impact of diabetes on the role of adenosine in regulation of retinal hemodynamics. Therefore, we examined how diabetes affects adenosine-induced vasodilation of retinal arterioles. Male Wistar rats were treated with streptozotocin (80 mg/kg, intraperitoneally), and experiments were performed 6-8 weeks later. Rats were treated with tetrodotoxin (50 microg/kg, intravenously [i.v.]) to eliminate any nerve activity and prevent movement of the eye and infused with methoxamine continuously to maintain adequate systemic circulation. Fundus images were captured with a digital camera that was equipped with a special objective lens, and diameters of retinal arterioles were measured. Adenosine increased diameters of retinal arterioles and decreased systemic blood pressure. These responses were significantly attenuated by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (30 mg/kg, i.v.) and the adenosine triphosphate-dependent K+ (K(ATP)) channel blocker glibenclamide (20 mg/kg, i.v.). The depressor responses to adenosine were reduced in diabetic rats, whereas diabetes did not alter vasodilation of retinal arterioles to adenosine. In contrast, both depressor response and vasodilation of retinal arteriole to acetylcholine were reduced in diabetic rats. The retinal vasodilator responses to adenosine and acetylcholine observed in diabetic rats were diminished by N(G)-nitro-L-arginine methyl ester. There were no differences in the responses to pinacidil, a K(ATP) channel opener, between the diabetic and nondiabetic rats. These results suggest that both the activation of nitric oxide synthase and opening of K(ATP) channels contribute to the vasodilator effects of adenosine in rats in vivo. However, diabetes has no significant impact on the vasodilation mediated by these mechanisms in

  7. Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

    PubMed

    Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2014-09-15

    Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists. PMID:25063794

  8. Airway hyperresponsiveness to adenosine induced by lipopolysaccharide in Brown Norway rats

    PubMed Central

    Tigani, B; Hannon, J P; Rondeau, C; Mazzoni, L; Fozard, J R

    2002-01-01

    We have explored the effects of bacterial endotoxin (lipopolysaccharide; LPS) on the response of the airways of Brown Norway (BN) rats to adenosine. Comparisons have been drawn with the effects on responses to methacholine and 5-hydroxytryptamine.In vehicle-challenged animals, adenosine, given i.v. was only a weak bronchoconstrictor. In contrast, 1 h following intratracheal administration of LPS, 0.3 mg kg−1, bronchoconstrictor responses to adenosine were markedly and selectively enhanced. At this time point, there were no significant changes in leukocyte numbers, eosinophil peroxidase and myeloperoxidase activities or protein concentrations in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, the sensitivity of the airways to both adenosine and methacholine was reduced relative to the earlier time point and there were substantial increases in each marker of inflammation in BAL fluid.The bronchoconstrictor response to adenosine was blocked selectively by methysergide, disodium cromoglycate and the broad-spectrum adenosine receptor antagonist, 8-SPT, but not by DPCPX or ZM 243185, selective antagonists for the A1 and A2A receptors, respectively.Thus, the response to adenosine augmented following LPS is mast cell mediated and involves a receptor which can be blocked by 8-SPT but not by selective A1 or A2A receptor antagonists. It thus bears similarity to the augmented response to adenosine induced by allergen challenge in actively sensitized BN rats. Exposure to LPS could be a factor along with allergen in determining the increased sensitivity of the airways of asthmatics to adenosine. PMID:11976275

  9. Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production.

    PubMed

    Clayton, Aled; Al-Taei, Saly; Webber, Jason; Mason, Malcolm D; Tabi, Zsuzsanna

    2011-07-15

    Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment. PMID:21677139

  10. Possible therapeutic benefits of adenosine-potentiating drugs in reducing age-related degenerative disease in dogs and cats.

    PubMed

    Scaramuzzi, R J; Baker, D J

    2003-10-01

    Adenosine is a ubiquitous, biologically important molecule that is a precursor of other biologically active molecules. It also is a component of some co-factors and has distinct physiological actions in its own right. Levels are maintained by synthesis from dietary precursors and re-cycling. The daily turnover of adenosine is very high. Adenosine can act either as a hormone by binding to adenosine receptors, four adenosine receptor subtypes have been identified, and as an intracellular modulator, after transport into the cell by membrane transporter proteins. One of the principal intracellular actions of adenosine is inhibition of the enzyme phosphodiesterase. Extracellular adenosine also has specific neuromodulatory actions on dopamine and glutamate. Selective and nonselective agonists and antagonists of adenosine are available. The tasks of developing, evaluating and exploiting the therapeutic potential of these compounds is still in its infancy. Adenosine has actions in the central nervous system (CNS), heart and vascular system, skeletal muscle and the immune system and the presence of receptors suggests potential actions in the gonads and other organs. Adenosine agonists improve tissue perfusion through actions on vascular smooth muscle and erythrocyte fluidity and they can be used to improve the quality of life in aged dogs. This article reviews the therapeutic potential of adenosine-potentiating drugs in the treatment of age-related conditions in companion animals, some of which may be exacerbated by castration or spaying at an early age. PMID:14633184

  11. Development of a luminescent G-quadruplex-selective iridium(III) complex for the label-free detection of adenosine

    PubMed Central

    Lu, Lihua; Zhong, Hai-Jing; He, Bingyong; Leung, Chung-Hang; Ma, Dik-Lung

    2016-01-01

    A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R2 = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis. PMID:26778273

  12. Development of a luminescent G-quadruplex-selective iridium(III) complex for the label-free detection of adenosine

    NASA Astrophysics Data System (ADS)

    Lu, Lihua; Zhong, Hai-Jing; He, Bingyong; Leung, Chung-Hang; Ma, Dik-Lung

    2016-01-01

    A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R2 = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis.

  13. A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Bustamante, Salvador; García-Sacristán, Albino; Orensanz, Luis M

    1999-01-01

    The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. In U46619 (10−7  M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10−6  M), adenosine and related analogues induced relaxations with the following potency order: 5′-N-ethylcarboxamidoadenosine (NECA)=5′-(N-cyclopropyl)-carboxamidoadenosine (CPCA)=2-chloroadenosine (2-CA)>adenosine>cyclopentyladenosine (CPA)=N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA)=2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680). Epithelium removal or incubation with indomethacin (3×10−6  M) and L-NG-nitroarginine (L-NOARG, 3×10−5  M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10−8 M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3×10−8  M and 10−7  M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10−5  M) and DPCPX (10−6  M), which block A1/A2-receptors, reduced such relaxations. In strips treated with guanethidine (10−5  M), atropine (10−7  M), L-NOARG (3×10−5  M) and indomethacin (3×10−6  M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10−4  M) induced contractions of preparations. 8-PT (10−5  M) increased both contractions. DPCPX (10−8  M), NECA (10−4  M), CPCA, (10−4  M) and 2-CA (10−4  M) did not alter the contractions to EFS. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids

  14. Association of adenosine receptor gene polymorphisms and in vivo adenosine A1 receptor binding in the human brain.

    PubMed

    Hohoff, Christa; Garibotto, Valentina; Elmenhorst, David; Baffa, Anna; Kroll, Tina; Hoffmann, Alana; Schwarte, Kathrin; Zhang, Weiqi; Arolt, Volker; Deckert, Jürgen; Bauer, Andreas

    2014-12-01

    Adenosine A1 receptors (A1ARs) and the interacting adenosine A2A receptors are implicated in neurological and psychiatric disorders. Variants within the corresponding genes ADORA1 and ADORA2A were shown associated with pathophysiologic alterations, particularly increased anxiety. It is unknown so far, if these variants might modulate the A1AR distribution and availability in different brain regions. In this pilot study, the influence of ADORA1 and ADORA2A variants on in vivo A1AR binding was assessed with the A1AR-selective positron emission tomography (PET) radioligand [(18)F]CPFPX in brains of healthy humans. Twenty-eight normal control subjects underwent PET procedures to calculate the binding potential BPND of [(18)F]CPFPX in cerebral regions and to assess ADORA1 and ADORA2A single nucleotide polymorphism (SNP) effects on regional BPND data. Our results revealed SNPs of both genes associated with [(18)F]CPFPX binding to the A1AR. The strongest effects that withstood even Bonferroni correction of multiple SNP testing were found in non-smoking subjects (N=22) for ADORA2A SNPs rs2236624 and rs5751876 (corr. Pall<0.05). SNP alleles previously identified at risk for increased anxiety like the rs5751876 T-allele corresponded to consistently higher A1AR availability in all brain regions. Our data indicate for the first time that variation of A1AR availability was associated with ADORA SNPs. The finding of increased A1AR availability in regions of the fear network, particularly in ADORA2A risk allele carriers, strongly warrants evaluation and replication in further studies including individuals with increased anxiety. PMID:24943643

  15. Passive targeting of ischemic-reperfused myocardium with adenosine-loaded silica nanoparticles

    PubMed Central

    Galagudza, Michael; Korolev, Dmitry; Postnov, Viktor; Naumisheva, Elena; Grigorova, Yulia; Uskov, Ivan; Shlyakhto, Eugene

    2012-01-01

    Pharmacological agents suggested for infarct size limitation have serious side effects when used at cardioprotective doses which hinders their translation into clinical practice. The solution to the problem might be direct delivery of cardioprotective drugs into ischemic-reperfused myocardium. In this study, we explored the potential of silica nanoparticles for passive delivery of adenosine, a prototype cardioprotective agent, into ischemic-reperfused heart tissue. In addition, the biodegradation of silica nanoparticles was studied both in vitro and in vivo. Immobilization of adenosine on the surface of silica nanoparticles resulted in enhancement of adenosine-mediated infarct size limitation in the rat model. Furthermore, the hypotensive effect of adenosine was attenuated after its adsorption on silica nanoparticles. We conclude that silica nanoparticles are biocompatible materials that might potentially be used as carriers for heart-targeted drug delivery. PMID:22619519

  16. 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.

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

    PubMed Central

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

    2014-01-01

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

  18. Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

    PubMed Central

    Newby, A C

    1980-01-01

    1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells. PMID:6249264

  19. Elevated Ecto-5’-nucleotidase-Mediated Increased Renal Adenosine Signaling Via A2B Adenosine Receptor Contributes to Chronic Hypertension

    PubMed Central

    Zhang, Weiru; Zhang, Yujin; Wang, Wei; Dai, Yingbo; Ning, Chen; Luo, Renna; Sun, Kaiqi; Glover, Louise; Grenz, Almut; Sun, Hong; Tao, Lijian; Zhang, Wenzheng; Colgan, Sean P.; Blackburn, Michael R.; Eltzschig, Holger K.; Kellems, Rodney E.; Xia, Yang

    2013-01-01

    Rationale Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood. Objective We sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression. Methods and Results Using high-throughput quantitative reverse-transcription polymerase chain reaction profiling, we discovered that the expression level of 5′-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II–infused mice, an animal model of hypertensive nephropathy. Genetic and pharmacological studies in mice revealed that elevated CD73-mediated excess renal adenosine preferentially induced A2B adenosine receptor (ADORA2B) production and that enhanced kidney ADORA2B signaling contributes to angiotensin II–induced hypertension. Similarly, in humans, we found that CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared with normal individuals and were further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 production in a hypoxia-inducible factor-α–dependent manner and underlies the pathogenesis of the disease. Finally, we revealed that hypoxia-inducible factor-α is an important factor responsible for angiotensin II–induced CD73 and ADORA2B expression at the transcriptional level. Conclusions Overall, our studies reveal that angiotensin II–induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling–mediated endothelin-1 induction in a hypoxia-inducible factor-α–dependent manner. The inhibition of excess adenosine

  20. Effect of adenosine on the formation of prostacyclin in the rabbit isolated heart.

    PubMed Central

    Karwatowska-Prokopczuk, E.; Ciabattoni, G.; Wennmalm, A.

    1988-01-01

    1. The effect of adenosine on cardiac biosynthesis of prostacyclin (PGI2) was investigated. Rabbit hearts were perfused according to Langendorff at controlled pressure (with or without theophylline), or at controlled flow. The content of 6-keto-prostaglandin1 alpha (6-keto-PGF1 alpha, metabolite of PGI2) in the coronary effluent under basal conditions and during infusion of adenosine was determined using a highly specific radioimmunoassay. 2. In other experiments, rings of rabbit aorta were incubated with or without adenosine and the production of 6-keto-PGF1 alpha was analysed as above. 3. Administration of adenosine (10 micron) to hearts perfused at controlled pressure increased the coronary flow by up to 38%. The peak concentration of 6-keto-PGF1 alpha in the effluent exceeded the control by 177% (P less than 0.01), and the total efflux of 6-keto-PGF1 alpha exceeded the control by 179% (P less than 0.001). Theophylline (50 micron) reduced these effects of adenosine by 23%, 43% and 51%, respectively, without influencing the uptake of adenosine into the heart. 4. When adenosine (1-10 micron) was administered to hearts perfused at controlled flow, a dose-dependent decrease in the perfusion pressure, by 27% and 44% respectively, was observed. In parallel, the resulting increase in 6-keto-PGF1 alpha efflux was considerably lower (49% (P less than 0.05) and 43% (NS), respectively). A similar decrease in perfusion pressure induced in the absence of adenosine decreased the efflux of 6-keto-PGF1 alpha, by 15% (P less than 0.01) and 32% (P less than 0.001), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3052678

  1. Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts

    PubMed Central

    Lee, Tsung-Ming; Chen, Wei-Ting; Yang, Chen-Chia; Lin, Shinn-Zong; Chang, Nen-Chung

    2015-01-01

    We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats. PMID:25388908

  2. Role of adenosine in the sympathetic activation produced by isometric exercise in humans.

    PubMed Central

    Costa, F; Biaggioni, I

    1994-01-01

    Isometric exercise increases sympathetic nerve activity and blood pressure. This exercise pressor reflex is partly mediated by metabolic products activating muscle afferents (metaboreceptors). Whereas adenosine is a known inhibitory neuromodulator, there is increasing evidence that it activates afferent nerves. We, therefore, examined the hypothesis that adenosine stimulates muscle afferents and participates in the exercise pressor reflex in healthy volunteers. Intraarterial administration of adenosine into the forearm, during venous occlusion to prevent systemic effects, mimicked the response to exercise, increasing muscle sympathetic nerve activity (MSNA, lower limb microneurography) and mean arterial blood pressure (MABP) at all doses studied (2, 3, and 4 mg). Heart rate increased only with the highest dose. Intrabrachial adenosine (4 mg) increased MSNA by 96 +/- 25% (n = 6, P < 0.01) and MABP by 12 +/- 3 mmHg (P < 0.01). Adenosine produced forearm discomfort, but equivalent painful stimuli (forearm ischemia and cold exposure) increased MSNA significantly less than adenosine. Furthermore, adenosine receptor antagonism with intrabrachial theophylline (1 microgram/ml forearm per min) blocked the increase in MSNA (92 +/- 15% vs. 28 +/- 6%, n = 7, P < 0.01) and MABP (38 +/- 6 vs. 27 +/- 4 mmHg, P = 0.01) produced by isometric handgrip (30% of maximal voluntary contraction) in the infused arm, but not the contralateral arm. Theophylline did not prevent the increase in heart rate produced by handgrip, a response mediated more by central command than muscle afferent activation. We propose that endogenous adenosine contributes to the activation of muscle afferents involved in the exercise pressor reflex in humans. PMID:8163667

  3. Role of adenosine A1 and A2A receptors in the alcohol withdrawal syndrome.

    PubMed

    Kaplan, G B; Bharmal, N H; Leite-Morris, K A; Adams, W R

    1999-10-01

    The role of adenosine receptor-mediated signaling was examined in the alcohol withdrawal syndrome. CD-1 mice received a liquid diet containing ethanol (6.7%, v/v) or a control liquid diet that were abruptly discontinued after 14 days of treatment. Mice consuming ethanol showed a progressive increase in signs of intoxication throughout the drinking period. Following abrupt discontinuation of ethanol diet, mice demonstrated reversible signs of handling-induced hyperexcitability that were maximal between 5-8 h. Withdrawing mice received treatment with adenosine receptor agonists at the onset of peak withdrawal (5.5 h) and withdrawal signs were blindly rated (during withdrawal hours 6 and 7). Adenosine A1-receptor agonist R-N6(phenylisopropyl)adenosine (0.15 and 0.3 mg/ kg) reduced withdrawal signs 0.5 and 1.5 h after drug administration in a dose-dependent fashion. Adenosine A2A-selective agonist 2-p-(2-carboxyethyl)phenylethyl-amino-5'-N-ethylcarboxamidoadenosine (0.3 mg/kg) reduced withdrawal signs at both time points. In ethanol-withdrawing mice, there were significant decreases in adenosine transporter sites in striatum without changes in cortex or cerebellum. In ethanol-withdrawing mice, there were no changes in adenosine A1 and A2A receptor concentrations in cortex, striatum, or cerebellum. There appears to be a role for adenosine A1 and A2A receptors in the treatment of the ethanol withdrawal syndrome. Published by Elsevier Science Inc. PMID:10548160

  4. Ticagrelor Does Not Inhibit Adenosine Transport at Relevant Concentrations: A Randomized Cross-Over Study in Healthy Subjects In Vivo

    PubMed Central

    Rongen, G. A.; van den Broek, P. H. H.; Bilos, A.; Donders, A. R. T.; Gomes, M. E.; Riksen, N. P.

    2015-01-01

    Background and Purpose In patients with myocardial infarction, ticagrelor reduces cardiovascular and sepsis-related mortality, and can cause dyspnea. It is suggested that this is caused by adenosine receptor stimulation, because in preclinical studies, ticagrelor blocks the nucleoside transporter and increases cellular ATP release. We now investigated the effects of ticagrelor on the adenosine system in humans in vivo. Experimental Approach In a double-blinded, placebo-controlled cross-over trial in 14 healthy subjects, we have tested whether ticagrelor (180 mg) affects adenosine- and dipyridamole-induced forearm vasodilation, as surrogates of nucleoside uptake inhibition and adenosine formation, respectively. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was measured. Primary endpoint was adenosine-induced vasodilation. Key Results Ticagrelor did not affect adenosine- or dipyridamole-induced forearm vasodilation. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was not affected by ticagrelor. In vitro, ticagrelor dose-dependently inhibited nucleoside uptake, but only at supra-physiological concentrations. Conclusion and Implications In conclusion, at relevant plasma concentration, ticagrelor does not affect adenosine transport, nor adenosine formation in healthy subjects. Therefore, it is unlikely that this mechanism is a relevant pleiotropic effect of ticagrelor. Trial Registration ClinicalTrials.gov NCT01996735 PMID:26509673

  5. Hypertonic NaCl enhances adenosine release and hormonal cAMP production in mouse thick ascending limb.

    PubMed

    Baudouin-Legros, M; Badou, A; Paulais, M; Hammet, M; Teulon, J

    1995-07-01

    Adenosine 3',5'-cyclic monophosphate (cAMP), accumulated in the presence of adenosine, was measured in medullary portions of mouse thick ascending limbs of Henle's loop, suspended either in classic extracellular buffer or in the presence of added NaCl. Under control conditions (140 mmol/l NaCl), adenosine (< 10(-5) mol/l) and N6-cyclohexyladenosine, an A1 adenosine receptor agonist, inhibit the cAMP accumulation induced by arginine vasopressin (AVP). On the other hand, high concentrations of adenosine and CGS-21680, an A2 adenosine receptor agonist, stimulate cAMP formation. Addition of NaCl (+300 mmol/l) to extracellular buffer stimulates the release of endogenous adenosine. It also enhances A2 receptor-induced cAMP accumulation but suppresses A1 receptor-mediated inhibition of adenylyl cyclase. This hypertonic NaCl medium also potentiates the stimulatory action of AVP on adenylyl cyclase. The modifications of tubular responses to both AVP and A1 and A2 agonists, brought about by hypertonic NaCl, were all inhibited by adenosine deaminase, thereby demonstrating the involvement of endogenous adenosine. Adenosine, the release and the effects of which are modulated by hypertonic NaCl, thus appears to act as an endogenous physiological modulator of kidney medulla function. PMID:7631823

  6. K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

    PubMed Central

    Heinrich, A; Andó, RD; Túri, G; Rózsa, B; Sperlágh, B

    2012-01-01

    BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K+, with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca2+-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K+-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels. LINKED ARTICLE This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x PMID:22394324

  7. Inhibition of Enterovirus 71 by Adenosine Analog NITD008

    PubMed Central

    Deng, Cheng-Lin; Yeo, Huimin; Ye, Han-Qing; Liu, Si-Qing; Shang, Bao-Di; Gong, Peng; Alonso, Sylvie

    2014-01-01

    ABSTRACT Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 μM and a 50% cytotoxic concentration of 119.97 μM. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment. IMPORTANCE Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of

  8. Cardiac endothelial transport and metabolism of adenosine and inosine

    PubMed Central

    Schwartz, Lisa M.; Bukowski, Thomas R.; Revkin, James H.; Bassingthwaighte, James B.

    2010-01-01

    The influence of transmembrane flux limitations on cellular metabolism of purine nucleosides was assessed in whole organ studies. Transcapillary transport of the purine nucleosides adenosine (Ado) and inosine (Ino) via paracellular diffusion through interendothelial clefts in parallel with carrier-mediated transendothelial fluxes was studied in isolated, Krebs-Henseleit-perfused rabbit and guinea pig hearts. After injection into coronary inflow, multiple-indicator dilution curves were obtained from coronary outflow for 90 s for 131I-labeled albumin (intravascular reference tracer), [3H]arabinofuranosyl hypoxanthine (AraH; extracellular reference tracer and nonreactive adenosine analog), and either [14C]Ado or [14C]Ino. Ado or Ino was separated from their degradative products, hypoxanthine, xanthine, and uric acid, in each outflow sample by HPLC and radioisotope counting. Ado and Ino, but not AraH, permeate the luminal membrane of endothelial cells via a saturable transporter with permeability-surface area product PSecl and also diffuse passively through interendothelial clefts with the same conductance (PSg) as AraH. These parallel conductances were estimated via fitting with an axially distributed, multi-pathway, four-region blood-tissue exchange model. PSg for AraH were ~4 and 2.5 ml · g−1 · min−1 in rabbits and guinea pigs, respectively. In contrast, transplasmalemmal conductances (endothelial PSecl) were ~0.2 ml · g−1 · min−1 for both Ado and Ino in rabbit hearts but ~2 ml · g−1 · min−1 in guinea pig hearts, an order of magnitude different. Purine nucleoside metabolism also differs between guinea pig and rabbit cardiac endothelium. In guinea pig heart, 50% of the tracer Ado bolus was retained, 35% was washed out as Ado, and 15% was lost as effluent metabolites; 25% of Ino was retained, 50% washed out, and 25% was lost as metabolites. In rabbit heart, 45% of Ado was retained and 5% lost as metabolites, and 7% of Ino was retained and 3% lost as

  9. AMID Mediates Adenosine-Induced Caspase-Independent HuH-7 Cell Apoptosis

    PubMed Central

    Yang, Dongqin; Yaguchi, Takahiro; Nagata, Tetsu; Gotoh, Akinobu; Dovat, Sinisa; Song, Chunhua; Nishizaki, Tomoyuki

    2011-01-01

    Background/Aims: The mechanism underlying extracellular adenosine-induced caspase-independent apoptosis in HuH-7 human hepatoma cells is not fully understood. The present study investigated the role for apoptosis-inducing factor (AIF)-homologous mitochondrion-associated inducer of death (AMID) in the pathway. Methods: To see the implication of AMID in adenosine-induced HuH-7 cell apoptosis, real-time reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescent cytochemistry, time-laps GFP monitoring, cell cycle analysis, flow cytometry, Western blotting, cell viability assay, and TUNEL staining were carried out. Results: Adenosine upregulated AMID expression in HuH-7 cells, and translocated AMID from the cytosol into the nucleus. Adenosine induced HuH-7 cell apoptosis, and the effect was further enhanced by overexpressing AMID. Adenosine-induced HuH-7 cell apoptosis, alternatively, was inhibited by knocking-down AMID. Conclusion: The results of the present study provide evidence for AMID as a critical factor for adenosine-induced caspase-independent HuH-7 cell apoptosis. PMID:21325820

  10. Synaptic mechanisms of adenosine A2A receptor-mediated hyperexcitability in the hippocampus.

    PubMed

    Rombo, Diogo M; Newton, Kathryn; Nissen, Wiebke; Badurek, Sylvia; Horn, Jacqueline M; Minichiello, Liliana; Jefferys, John G R; Sebastiao, Ana M; Lamsa, Karri P

    2015-05-01

    Adenosine inhibits excitatory neurons widely in the brain through adenosine A1 receptor, but activation of adenosine A2A receptor (A2A R) has an opposite effect promoting discharge in neuronal networks. In the hippocampus A2A R expression level is low, and the receptor's effect on identified neuronal circuits is unknown. Using optogenetic afferent stimulation and whole-cell recording from identified postsynaptic neurons we show that A2A R facilitates excitatory glutamatergic Schaffer collateral synapses to CA1 pyramidal cells, but not to GABAergic inhibitory interneurons. In addition, A2A R enhances GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. Adenosine A2A R has no direct modulatory effect on GABAergic synapses to pyramidal cells. As a result adenosine A2A R activation alters the synaptic excitation - inhibition balance in the CA1 area resulting in increased pyramidal cell discharge to glutamatergic Schaffer collateral stimulation. In line with this, we show that A2A R promotes synchronous pyramidal cell firing in hyperexcitable conditions where extracellular potassium is elevated or following high-frequency electrical stimulation. Our results revealed selective synapse- and cell type specific adenosine A2A R effects in hippocampal CA1 area. The uncovered mechanisms help our understanding of A2A R's facilitatory effect on cortical network activity. PMID:25402014

  11. Unraveling the role of adenosine in remote ischemic preconditioning-induced cardioprotection.

    PubMed

    Randhawa, Puneet Kaur; Jaggi, Amteshwar Singh

    2016-06-15

    Remote ischemic preconditioning (RIPC) induced by alternate cycles of preconditioning ischemia and reperfusion protects the heart against sustained ischemia-reperfusion-induced injury. This technique has been translated to clinical levels in patients undergoing various surgical interventions including coronary artery bypass graft surgery, abdominal aortic aneurysm repair, percutaneous coronary intervention and heart valve surgery. Adenosine is a master regulator of energy metabolism and reduces myocardial ischemia-reperfusion-induced injury. Furthermore, adenosine is a critical trigger as well as a mediator in RIPC-induced cardioprotection and scientists have demonstrated the role of adenosine by showing an increase in its levels in the systemic circulation during RIPC delivery. Furthermore, the blockade of cardioprotective effects of RIPC in the presence of specific adenosine receptor blockers and transgenic animals with targeted ablation of A1 receptors has also demonstrated its critical role in RIPC. The studies have shown that adenosine may elicit cardioprotection via activation of neurogenic pathway. The present review describes the possible role and mechanism of adenosine in mediating RIPC-induced cardioprotection. PMID:27157518

  12. Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells

    SciTech Connect

    Cronstein, B.N.; Eberle, M.A.; Levin, R.I. ); Gruber, H.E. )

    1991-03-15

    Although commonly used to control a variety of inflammatory diseases, the mechanism of action of a low dose of methotrexate remains a mystery. Methotrexate accumulates intracellularly where it may interfere with purine metabolism. Therefore, the authors determined whether a 48-hr pretreatment with methotrexate affected adenosine release from ({sup 14}C)adenine-labeled human fibroblasts and umbilical vein endothelial cells. Methotrexate significantly increased adenosine release by fibroblasts. The effect of methotrexate on adenosine release was not due to cytotoxicity since cells treated with maximal concentrations of methotrexate took up ({sup 14}C)adenine and released {sup 14}C-labeled purine (a measure of cell injury) in a manner identical to control cells. Methotrexate treatment of fibroblasts dramatically inhibited adherence to fibroblasts by both unstimulated neutrophils and stimulated neutrophils. One hypothesis that explains the effect of methotrexate on adenosine release is that, by inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, methotrexate induces the accumulation of AICAR, the nucleoside precursor of which has previously been shown to cause adenosine release from ischemic cardiac tissue. The observation that the antiinflammatory actions of methotrexate are due to the capacity of methotrexate to induce adenosine release may form the basis for the development of an additional class of antiinflammatory drugs.

  13. Functional proteomics of adenosine triphosphatase system in the rat striatum during aging☆

    PubMed Central

    Villa, Roberto Federico; Ferrari, Federica; Gorini, Antonella

    2012-01-01

    The maximum rates of adenosine triphosphatase (ATPase) systems related to energy consumption were systematically evaluated in synaptic plasma membranes isolated from the striata of male Wistar rats aged 2, 6, 12, 18, and 24 months, because of their key role in presynaptic nerve ending homeostasis. The foll