Sample records for adenosine triphosphate hydrolysis

  1. 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.

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

    PubMed Central

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

    2014-01-01

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

  3. 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.

  4. The Role of Extracellular Adenosine Triphosphate in Ischemic Organ Injury.

    PubMed

    Zhao, Hailin; Kilgas, Susan; Alam, Azeem; Eguchi, Shiori; Ma, Daqing

    2016-05-01

    Ischemic tissue injury contributes to significant morbidity and mortality and is implicated in a range of pathologic conditions, including but not limited to myocardial infarction, ischemic stroke, and acute kidney injury. The associated reperfusion phase is responsible for the activation of the innate and adaptive immune system, further accentuating inflammation. Adenosine triphosphate molecule has been implicated in various ischemic conditions, including stroke and myocardial infarction. Adenosine triphosphate is a well-defined intracellular energy transfer and is commonly referred to as the body's "energy currency." However, Laboratory studies have demonstrated that extracellular adenosine triphosphate has the ability to initiate inflammation and is therefore referred to as a damage-associated molecular pattern. Purinergic receptors-dependent signaling, proinflammatory cytokine release, increased Ca influx into cells, and subsequent apoptosis have been shown to form a common underlying extracellular adenosine triphosphate molecular mechanism in ischemic organ injury. In this review, we aim to discuss the molecular mechanisms behind adenosine triphosphate-mediated ischemic tissue injury and evaluate the role of extracellular adenosine triphosphate in ischemic injury in specific organs, in order to provide a greater understanding of the pathophysiology of this complex process. We also appraise potential future therapeutic strategies to limit damage in various organs, including the heart, brain, kidneys, and lungs.

  5. Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.

    PubMed

    Li, Xiaoou; Kondo, Yutaka; Bao, Yi; Staudenmaier, Laura; Lee, Albert; Zhang, Jingping; Ledderose, Carola; Junger, Wolfgang G

    2017-01-01

    Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis. Prospective randomized animal investigation and in vitro studies. Preclinical academic research laboratory. Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies. Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis. Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis. Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses

  6. Does adenosine triphosphate released into voided urodynamic fluid contribute to urgency signaling in women with bladder dysfunction?

    PubMed

    Cheng, Ying; Mansfield, Kylie J; Allen, Wendy; Walsh, Colin A; Burcher, Elizabeth; Moore, Kate H

    2010-03-01

    Adenosine triphosphate released from urothelium during stretch stimulates afferent nerves and conveys information on bladder fullness. We measured adenosine triphosphate released during cystometric bladder filling in women with idiopathic detrusor overactivity and stress incontinence (controls), and assessed whether the level of released adenosine triphosphate is related to cystometric parameters. Routine cystometry was done in 51 controls and 48 women with detrusor overactivity who were 28 to 87 years old. Voided urodynamic fluid was collected and stored at -30 C. Adenosine triphosphate was measured by a bioluminescence assay. Adenosine triphosphate levels were similar in voided urodynamic fluid of controls and patients with detrusor overactivity (p = 0.79). A significant inverse correlation was seen between adenosine triphosphate and maximal cystometric capacity in controls (p = 0.013), and between voided volume and adenosine triphosphate in controls (p = 0.015) and detrusor overactivity cases (p = 0.019). A significant correlation between first desire to void and adenosine triphosphate was also noted in detrusor overactivity cases (p = 0.033) but not in controls (p = 0.58). No correlation was seen between adenosine triphosphate and detrusor pressure during filling or voiding. Adenosine triphosphate measurement in voided urodynamic fluid is a novel approach to understanding signals that may contribute to the urgency sensation (a sudden compelling desire to pass urine). The inverse correlation between adenosine triphosphate in voided urodynamic fluid and first desire to void suggests that adenosine triphosphate has a role in modulating the early filling sensation in patients with detrusor overactivity. 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  7. Immune monitoring with a lymphocyte adenosine triphosphate assay in kidney transplant recipients treated with a calcineurin inhibitor.

    PubMed

    Sugiyama, Kentaro; Tsukaguchi, Mahoto; Toyama, Akira; Satoh, Hiroshi; Saito, Kazuhide; Nakagawa, Yuki; Takahashi, Kota; Tanaka, Sachiko; Onda, Kenji; Hirano, Toshihiko

    2014-06-01

    The adenosine triphosphate assay using peripheral lymphocytes may be useful to evaluate the risks of acute rejection and infection in kidney transplant patients. We used the adenosine triphosphate assay to evaluate differences between recipients who were treated with cyclosporine- or tacrolimus-based immunosuppressive therapy. Adenosine triphosphate levels were measured in peripheral CD4+ cells before and after transplant and were correlated with clinical outcomes in 45 kidney transplant recipients. These recipients received immunosuppressive therapy with either cyclosporine (23 patients) or tacrolimus (22 patients). Adenosine triphosphate levels were significantly lower in the cyclosporine- than tacrolimus-based therapy groups from 2 to 6 weeks after transplant. Adenosine triphosphate levels were similar between these groups before and 1 week after transplant. The frequency of cytomegalovirus infection was greater in the recipients who received cyclosporine (17 patients [74%]) than tacrolimus (6 patients [27%]; P ≦ .003). The frequency of acute rejection episodes was similar between the cyclosporine and tacrolimus groups. These observations suggest that cyclosporine-based immunosuppressive therapy causes excessive immunosuppression compared with tacrolimus-based therapy, evidenced by the lymphocyte adenosine triphosphate levels. The adenosine triphosphate assay using peripheral CD4+ cells may be a useful method for predicting the occurrence of cytomegalovirus infections in kidney transplant recipients.

  8. 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.

  9. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  10. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  11. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  12. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  13. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  14. Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice.

    PubMed

    Bayliss, Jill; Delarosa, Sara; Wu, Jianfeng; Peterson, Jonathan R; Eboda, Oluwatobi N; Su, Grace L; Hemmila, Mark; Krebsbach, Paul H; Cederna, Paul S; Wang, Stewart C; Xi, Chuanwu; Levi, Benjamin

    2014-01-01

    Extracellular adenosine triphosphate (ATP), present in thermally injured tissue, modulates the inflammatory response and causes significant tissue damage. The authors hypothesize that neutrophil infiltration and ensuing tissue necrosis would be mitigated by removing ATP-dependent signaling at the burn site. Mice were subjected to 30% TBSA partial-thickness scald burn by dorsal skin immersion in a water bath at 60 or 20°C (nonburn controls). In the treatment arm, an ATP hydrolyzing enzyme, apyrase, was applied directly to the site immediately after injury. Skin was harvested after 24 hours and 5 days for hematoxylin and eosin stain, elastase, and Ki-67 staining. Tumor necrosis factor (TNF)-α and interferon (IFN)-β expression were measured through quantitative real-time polymerase chain reaction. At 24 hours, the amount of neutrophil infiltration was different between the burn and burn + apyrase groups (P < .001). Necrosis was less extensive in the apyrase group when compared with the burn group at 24 hours and 5 days. TNF-α and IFN-β expression at 24 hours in the apyrase group was lower than in the burn group (P < .05). However, Ki-67 signaling was not significantly different among the groups. The results of this study support the role of extracellular ATP in neutrophil activity. The authors demonstrate that ATP hydrolysis at the burn site allays the neutrophil response to thermal injury and reduces tissue necrosis. This decrease in inflammation and tissue necrosis is at least partially because of TNF-α and IFN-β signaling. Apyrase could be used as topical inflammatory regulators to quell the injury caused by inflammation.

  15. Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

    PubMed Central

    2011-01-01

    Background Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. Results NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. Conclusion Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses. PMID:22035583

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

    PubMed

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

    2014-08-01

    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. 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. 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.

  17. Extracellular adenosine triphosphate affects systemic and kidney immune cell populations in pregnant rats.

    PubMed

    Spaans, Floor; Melgert, Barbro N; Borghuis, Theo; Klok, Pieter A; de Vos, Paul; Bakker, Winston W; van Goor, Harry; Faas, Marijke M

    2014-09-01

    Changes in the systemic immune response are found in preeclampsia. This may be related to high extracellular adenosine triphosphate (ATP) levels. The question arose whether ATP could affect immune responses in pregnancy. Previously, we investigated whether ATP affected monocyte activation and subpopulations. Here, we investigated ATP-induced changes in other immune cell populations in pregnant rats, systemically and in the kidney, an affected organ in preeclampsia. Using flow cytometry or immunohistochemistry, blood and kidney leukocytes were studied in pregnant and non-pregnant rats at different intervals after ATP or saline infusion. Adenosine triphosphate (ATP) infusion induced increased peripheral blood non-classical monocytes and decreased T lymphocyte subsets in pregnant rats only, higher glomerular macrophage and T lymphocyte numbers in non-pregnant animals 1 day after infusion, and higher glomerular macrophage numbers in pregnant rats 6 days after infusion. Adenosine triphosphate (ATP) infusion in pregnant rats induced a pregnancy-specific inflammatory response. Increased ATP levels could potentially contribute to development of the inflammatory response of preeclampsia. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Changes in Adenosine Triphosphate and Nitric Oxide in the Urothelium of Patients with Benign Prostatic Hyperplasia and Detrusor Underactivity.

    PubMed

    Cho, Kang Jun; Koh, Jun Sung; Choi, Jinbong; Kim, Joon Chul

    2017-12-01

    We investigated changes in the levels of adenosine triphosphate and nitric oxide in the urothelium of men with detrusor underactivity and benign prostatic hyperplasia. We prospectively enrolled in study 30 men who planned to undergo surgical treatment for benign prostatic hyperplasia. The 15 patients with a bladder contractility index less than 100 were assigned to the detrusor underactivity group while the 15 with a bladder contractility index more than 100 were assigned to the no detrusor underactivity group. Bladder mucosal specimens were collected at surgical prostate resection, and adenosine triphosphate and endothelial nitric oxide synthase were analyzed in these specimens. The levels of adenosine triphosphate and endothelial nitric oxide synthase were compared between the 2 groups. The correlation of urodynamic parameters with adenosine triphosphate and endothelial nitric oxide synthase was assessed in all patients. Mean ± SEM endothelial nitric oxide synthase did not significantly differ between the detrusor underactivity and no underactivity groups (3.393 ± 0.969 vs 1.941 ± 0.377 IU/ml, p = 0.247). However, the mean level of adenosine triphosphate in the detrusor underactivity group was significantly lower than in the no detrusor underactivity group (1.289 ± 0.320 vs 9.262 ± 3.285 pmol, p = 0.011). In addition, in all patients adenosine triphosphate positively correlated with the bladder contractility index (r = 0.478, p = 0.018) and with detrusor pressure on maximal flow (r = 0.411, p = 0.046). Adenosine triphosphate was significantly decreased in the urothelium in men with detrusor underactivity and benign prostatic hyperplasia, reflecting the change in detrusor function. Copyright © 2017 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  19. The effects of noradrenaline and adenosine 5'-triphosphate on polyphosphoinositide and phosphatidylcholine hydrolysis in arterial smooth muscle.

    PubMed Central

    Nally, J. E.; Muir, T. C.; Guild, S. B.

    1992-01-01

    1. The effects of noradrenaline and alpha,beta,methylene adenosine 5'-triphosphate (alpha,beta,methylene ATP) on polyphosphoinositide metabolism, phosphatidylcholine hydrolysis and contraction in rabbit saphenous arteries were investigated. The effect of noradrenaline upon polyphosphoinositide metabolism was also investigated in the rat tail artery. 2. Noradrenaline (10(-7)-10(-4) M) evoked a concentration-dependent increase in total inositol phosphate accumulation in the rat tail but not in the rabbit saphenous artery. Propranolol (3 x 10(-6) M) did not alter this result in the rabbit saphenous artery. In addition, alpha,beta,methylene ATP (10(-6) M) significantly increased total inositol phosphate accumulation in the rabbit saphenous artery, while potassium chloride (8 x 10(-2) M) was ineffective. 3. Phorbol 1,2-myristate 1,3-acetate (3 x 10(-8) M) enhanced noradrenaline (10(-2)-10(-4) M)-evoked contractions in rabbit saphenous artery. The contractile responses to potassium chloride (1- 16 x 10(-2) M) in tissues treated with 6-hydroxydopamine (5 x 10(-4) M), in vitro, were unaffected by these concentrations of the phorbol ester. 4. Noradrenaline (10(-6)-10(-4) M) evoked a concentration-dependent increase in the levels of choline and choline phosphate, but not in those of glycerophosphocholine, in the rabbit saphenous artery. Choline levels increased significantly over the first 15-30 s then declined to control levels within 2 min of addition of noradrenaline (10(-5) M). A smaller initial rise in choline phosphate levels (15-30 s) was followed by a larger secondary rise at 2-4 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1327389

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

    PubMed Central

    Fedan, J. S.

    1987-01-01

    The effects of incubating the guinea-pig isolated vas deferens in the presence of adenine nucleotides (adenosine triphosphate, ATP; adenosine diphosphate, ADP; and adenosine monophosphate, AMP), or in the presence of their phosphorothioate analogues (adenosine 5'-O-(3-thiotriphosphate), ATP gamma S; adenosine 5'-O-(2-thiodiphosphate), ADP beta S; and adenosine 5'-monophosphorothioate, AMP alpha S), on contractile responses to ATP were compared. After challenge with a low (1 microM) or high (300 microM) concentration of ATP to obtain control responses, one vas deferens of a pair was incubated for 5 min with one of the adenine nucleotides, while the contralateral preparation was incubated with the corresponding phosphorothioate analogue. At the conclusion of the incubation the preparations were challenged again with ATP. Incubation with AMP or AMP alpha S resulted in a transient potentiation of responses to 1 microM and 300 microM ATP. The potentiation following incubation with AMP alpha S was larger than that produced by AMP. After incubation with ADP, ADP beta S, ATP and ATP gamma S, responses to 1 microM ATP were decreased, while those to 300 microM ATP were unaffected. Thus, incubation with AMP and AMP alpha S results in potentiation, rather than inhibition, of ATP-induced responses. On the other hand, 5'-diphosphate, 5'-triphosphate, 5'-O-(2-thiodiphosphate) and 5'-O-(3-thiotriphosphate) moieties on adenosine have no effect or cause autoinhibition. These results indicate that AMP exerts a potentiating effect on reactivity to exogenous ATP. AMP arising from the enzymatic degradation of ATP might modulate the level of response to ATP released endogenously as a cotransmitter. PMID:3038248

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

    PubMed

    Butler, Stephen J

    2014-11-24

    Two tripodal fluorescent probes Zn⋅L(1,2) have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn⋅L(1) allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of several competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

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

    2013-11-08

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

  3. 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.

  4. Imaging Adenosine Triphosphate (ATP)

    PubMed Central

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-01-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provides valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific for ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies that are available to visualize ATP in living cells and identify areas where new tools and approaches are needed to expand our capabilities. PMID:27638696

  5. Imaging Adenosine Triphosphate (ATP).

    PubMed

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-08-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provide valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to the organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific to ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies available for visualizing ATP in living cells, and identify areas where new tools and approaches are needed to expand our capabilities. © 2016 Marine Biological Laboratory.

  6. Hydrolysis of Extracellular ATP by Ectonucleoside Triphosphate Diphosphohydrolase (ENTPD) Establishes the Set Point for Fibrotic Activity of Cardiac Fibroblasts*

    PubMed Central

    Lu, David; Insel, Paul A.

    2013-01-01

    The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs. PMID:23677997

  7. 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.

  8. Paul D. Boyer, Adenosine Triphosphate (ATP), and the Binding Change

    Science.gov Websites

    -- October 1975, DOE Technical Report, 1975 A Perspective of the Binding Change Mechanism for ATP Synthesis Reports, Vol. 18, No. 3, 1998 ATP Synthesis and the Binding Change Mechanism: The Work of Paul D. Boyer Mechanism of ATP Synthesis Additional Web Pages: Adenosine Triphosphate: The Energy Currency of Life Paul D

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

    PubMed

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

    2013-09-17

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

  10. Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: role of adenosine receptors.

    PubMed

    Maldonado, Claudio; Pushpakumar, Sathnur B; Perez-Abadia, Gustavo; Arumugam, Sengodagounder; Lane, Andrew N

    2013-05-01

    Ischemia-reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. The extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. PCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5'-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. Exogenous ATP administration to ischemic skeletal

  11. 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

  12. Optical Aptasensors for Adenosine Triphosphate.

    PubMed

    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.

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

    PubMed

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

    2015-01-30

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

  14. How Mg2+ ions lower the SN2@P barrier in enzymatic triphosphate hydrolysis.

    PubMed

    van Bochove, Marc A; Roos, Goedele; Fonseca Guerra, Célia; Hamlin, Trevor A; Bickelhaupt, F Matthias

    2018-04-03

    Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: (a) weakening of the leaving-group bond, thereby decreasing activation strain; and (b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrate, thereby strengthening the interaction with the nucleophile.

  15. A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis.

    PubMed

    Jenkins, R H; Tuma, R; Juuti, J T; Bamford, D H; Thomas, G J

    1999-01-01

    A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2017-06-01

    Biochemical and microbial changes after harvest strongly affect the final quality and shelf life of fish and fish products. In this study, the role of microbes in the degradation of adenosine triphosphate (ATP), and the origin of adenosine monophosphate deaminase (AMPD) and acid phosphatase (ACP) in common carp fillets during different stages of chilled storage (at 4°C) were investigated. The content of ATP, ADP, AMP, IMP, HxR, and Hx, the activity of AMPD and ACP, and the total count of viable, Aeromonas, Pseudomonas, H 2 S-producing bacteria, and lactic acid bacteria were examined. Results indicated that the population of microbial communities in control samples increased with storage time, and Pseudomonas peaked on the 10th day of storage. Changes in AMPD activity were less related to the abundance of microbes during the entire storage period. However, ACP was derived from both fish muscle and microbial secretion during the middle and late stages of storage. Degradation of ATP to IMP was not affected by spoilage bacteria, but the hydrolysis of IMP, and the transformation of HxR to Hx was affected considerably by the spoilage bacteria. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2014-12-10

    Adenosine triphosphate (ATP) is a molecule with a fascinating variety of intracellular and extracellular biological functions that go far beyond energy metabolism. Due to its limited passive diffusion through biological membranes, controlled release systems may allow to interact with ATP-mediated extracellular processes. In this study, two release systems were explored to evaluate the capacity for either long-term or short-term release: (i) Poly[(rac-lactide)-co-glycolide] (PLGA) implant rods were capable of ATP release over days to weeks, depending on the PLGA molecular weight and end-group capping, but were also associated with partial hydrolytic degradation of ATP to ADP and AMP, but not adenosine. (ii) Thermosensitive methylcellulose hydrogels with a gelation occurring at body temperature allowed combining adjustable loading levels and the capacity for injection, with injection forces less than 50N even for small 27G needles. Finally, a first in vitro study illustrated purinergic-triggered response of primary murine microglia to ATP released from hydrogels, demonstrating the potential relevance for biomedical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. 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 acdurately 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 -30°C were stable for months.

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

    PubMed

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

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

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

    PubMed Central

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

    1971-01-01

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

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

    PubMed

    Tiret, Brice; Brouillet, Emmanuel; Valette, Julien

    2016-09-01

    With the increased spectral resolution made possible at high fields, a second, smaller inorganic phosphate resonance can be resolved on (31)P magnetic resonance spectra in the rat brain. Saturation transfer was used to estimate de novo adenosine triphosphate synthesis reaction rate. While the main inorganic phosphate pool is used by adenosine triphosphate synthase, the second pool is inactive for this reaction. Accounting for this new pool may not only help us understand (31)P magnetic resonance spectroscopy metabolic profiles better but also better quantify adenosine triphosphate synthesis. © The Author(s) 2016.

  3. 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.

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

    NASA Astrophysics Data System (ADS)

    Hou, Faju; Miao, Yanhong; Jiang, Chongqiu

    2005-10-01

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

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

    PubMed

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

    2015-02-01

    This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5'-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the determination of adenosine in urine. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Chida, Junji; Kido, Hiroshi

    2014-01-01

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

  8. Vasodilatory responsiveness to adenosine triphosphate in ageing humans.

    PubMed

    Kirby, Brett S; Crecelius, Anne R; Voyles, Wyatt F; Dinenno, Frank A

    2010-10-15

    Endothelium-dependent vasodilatation is reduced with advancing age in humans, as evidenced by blunted vasodilator responsiveness to acetylcholine (ACh). Circulating adenosine triphosphate (ATP) has been implicated in the control of skeletal muscle vascular tone during mismatches in oxygen delivery and demand (e.g. exercise) via binding to purinergic receptors (P2Y) on the endothelium evoking subsequent vasodilatation, and ageing is typically associated with reductions in muscle blood flow under such conditions. Therefore, we tested the hypothesis that ATP-mediated vasodilatation is impaired with age in healthy humans. We measured forearm blood flow (venous occlusion plethysmography) and calculated vascular conductance (FVC) responses to local intra-arterial infusions of ACh, ATP, and sodium nitroprusside (SNP) before and during ascorbic acid (AA) infusion in 13 young and 13 older adults. The peak increase in FVC to ACh was significantly impaired in older compared with young adults (262 ± 71% vs. 618 ± 97%; P < 0.05), and this difference was abolished during AA infusion (510 ± 82% vs. 556 ± 71%; not significant, NS). In contrast, peak FVC responses were not different between older and young adults to either ATP (675 ± 105% vs. 734 ± 126%) or SNP (1116 ± 111% vs. 1138 ± 148%) and AA infusion did not alter these responses in either age group (both NS). In another group of six young and six older adults, we determined whether vasodilator responses to adenosine and ATP were influenced by P1-receptor blockade via aminophylline. The peak FVC responses to adenosine were not different in young (350 ± 65%) versus older adults (360 ± 80%), and aminophylline blunted these responses by ∼50% in both groups. The peak FVC responses to ATP were again not different in young and older adults, and aminophylline did not impact the vasodilatation in either group. Thus, in contrast to the observed impairments in ACh responses, the vasodilatory response to exogenous ATP is not

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

    PubMed

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

    2015-12-07

    There is enormous potential in combining the capabilities of the biological and the solid state to create hybrid engineered systems. While there have been recent efforts to harness power from naturally occurring potentials in living systems in plants and animals to power complementary metal-oxide-semiconductor integrated circuits, here we report the first successful effort to isolate the energetics of an electrogenic ion pump in an engineered in vitro environment to power such an artificial system. An integrated circuit is powered by adenosine triphosphate through the action of Na(+)/K(+) adenosine triphosphatases in an integrated in vitro lipid bilayer membrane. The ion pumps (active in the membrane at numbers exceeding 2 × 10(6) mm(-2)) are able to sustain a short-circuit current of 32.6 pA mm(-2) and an open-circuit voltage of 78 mV, providing for a maximum power transfer of 1.27 pW mm(-2) from a single bilayer. Two series-stacked bilayers provide a voltage sufficient to operate an integrated circuit with a conversion efficiency of chemical to electrical energy of 14.9%.

  10. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Effects of protonation on the hydrolysis of triphosphate in vacuum and the implications for catalysis by nucleotide hydrolyzing enzymes.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-06-29

    Nucleoside triphosphate (NTP) hydrolysis is a key reaction in biology. It involves breaking two very stable bonds (one P-O bond and one O-H bond of water), in either a concurrent or a sequential way. Here, we systematically examine how protonation of the triphosphate affects the mechanism of hydrolysis. The hydrolysis reaction of methyl triphosphate in vacuum is computed with protons in various numbers and position on the three phosphate groups. Protonation is seen to have a strong catalytic effect, with the reaction mechanism depending highly on the protonation pattern. This dependence is apparently complicated, but is shown to obey a well-defined set of rules: Protonation of the α- and β-phosphate groups favors a sequential hydrolysis mechanism, whereas γ-protonation favors a concurrent mechanism, the two effects competing with each other in cases of simultaneous protonation. The rate-limiting step is always the breakup of the water molecule while it attacks the γ-phosphorus, and its barrier is lowered by γ-protonation. This step has significantly lower barriers in the sequential reactions, because the dissociated γ-metaphosphate intermediate (P γ O 3 - ) is a much better target for water attack than the un-dissociated γ-phosphate (-P γ O 4 2- ). The simple chemical logic behind these rules helps to better understand the catalytic strategy used by NTPase enzymes, as illustrated here for the catalytic pocket of myosin. A set of rules was determined that describes how protonating the phosphate groups affects the hydrolysis mechanism of methyl triphosphate: Protonation of the α- and/or β- phosphate groups promotes a sequential mechanism in which P-O bond breaking precedes the breakup of the attacking water, whereas protonation of the γ-phosphate promotes a concurrent mechanism and lowers the rate-limiting barrier of water breakup. The role played by individual protein residues in the catalytic pocket of triphosphate hydrolysing enzymes can be assigned

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

    PubMed

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

    2012-11-21

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

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

    PubMed

    Rocha, Jeová Nina

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

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

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

    PubMed

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

    2012-09-21

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

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

    PubMed

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

    2013-09-07

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

  17. Noble Gas Xenon Is a Novel Adenosine Triphosphate-sensitive Potassium Channel Opener

    PubMed Central

    Bantel, Carsten; Maze, Mervyn; Trapp, Stefan

    2010-01-01

    Background Adenosine triphosphate-sensitive potassium (KATP) channels in brain are involved in neuroprotective mechanisms. Pharmacologic activation of these channels is seen as beneficial, but clinical exploitation by using classic K+ channel openers is hampered by their inability to cross the blood–brain barrier. This is different with the inhalational anesthetic xenon, which recently has been suggested to activate KATP channels; it partitions freely into the brain. Methods To evaluate the type and mechanism of interaction of xenon with neuronal-type KATP channels, these channels, consisting of Kir6.2 pore-forming subunits and sulfonylurea receptor-1 regulatory subunits, were expressed in HEK293 cells and whole cell, and excised patch-clamp recordings were performed. Results Xenon, in contrast to classic KATP channel openers, acted directly on the Kir6.2 subunit of the channel. It had no effect on the closely related, adenosine triphosphate (ATP)-regulated Kir1.1 channel and failed to activate an ATP-insensitive mutant version of Kir6.2. Furthermore, concentration–inhibition curves for ATP obtained from inside-out patches in the absence or presence of 80% xenon revealed that xenon reduced the sensitivity of the KATP channel to ATP. This was reflected in an approximately fourfold shift of the concentration causing half-maximal inhibition (IC50) from 26 ± 4 to 96 ± 6 μm. Conclusions Xenon represents a novel KATP channel opener that increases KATP currents independently of the sulfonylurea receptor-1 subunit by reducing ATP inhibition of the channel. Through this action and by its ability to readily partition across the blood–brain barrier, xenon has considerable potential in clinical settings of neuronal injury, including stroke. PMID:20179498

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

    PubMed

    Zhao, Qiang; Lv, Qin; Wang, Hailin

    2015-08-15

    We previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMR-labeled aptamers enabled direct FA sensing ATP with a detection limit of 1 µM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

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

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

    PubMed

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

    2015-10-01

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

  1. Snapshots of the maltose transporter during ATP hydrolysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oldham, Michael L.; Chen, Jue

    2011-12-05

    ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

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

    PubMed

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

    2015-03-09

    This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD. Copyright © 2015 John Wiley & Sons, Inc.

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

    PubMed

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

    2017-11-01

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

  4. The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

    PubMed Central

    Goldfarb, P. S. G.; Rodnight, R.

    1970-01-01

    1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed

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

    PubMed

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

    2016-04-15

    Adenosine triphosphate (ATP) is the most direct source of energy in organisms. This study is the first to demonstrate that ATP-aptamer complexes provide greater protection for unmodified gold nanoparticles (AuNPs) against salt-induced aggregation than either aptamer or ATP alone. This protective effect was confirmed using transmission electron microscopy, dynamic light scattering, Zeta potential measurement, and fluorescence polarization techniques. Utilizing controlled particle aggregation/dispersion as a gauge, a sensitive and selective aptasensor for colorimetric detection of ATP was developed using ATP-binding aptamers as the identification element and unmodified AuNPs as the probe. This aptasensor exhibited a good linear relationship between the absorbance and the logarithm concentration of ATP within a 50-1000 nM range. ATP analogs such as guanosine triphosphate, uridine triphosphate and cytidine triphosphate resulted in little or no interference in the determination of ATP. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2015-03-03

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

  7. Application of adenosine triphosphate-driven bioluminescence for quantification of plaque bacteria and assessment of oral hygiene in children.

    PubMed

    Fazilat, Shahram; Sauerwein, Rebecca; McLeod, Jennifer; Finlayson, Tyler; Adam, Emilia; Engle, John; Gagneja, Prashant; Maier, Tom; Machida, Curtis A

    2010-01-01

    Dentistry has undergone a shift in caries management toward prevention and improved oral hygiene and diagnosis. Caries prevention now represents one of the most important aspects of modern dental practice. The purpose of this cross-sectional study was to demonstrate the use of adenosine triphosphate- (ATP-) driven bioluminescence as an innovative tool for the rapid chairside enumeration of oral bacteria (including plague streptococci) and assessment of oral hygiene and caries risk. Thirty-three pediatric patients (7- to 12-year-old males and females) were examined, and plague specimens, in addition to stimulated saliva, were collected from representative teeth within each quadrant. Oral specimens (n=150 specimens) were assessed by plating on enriched and selective agars, to enumerate total bacteria and streptococci, and subjected to adenosine triphosphate- (ATP-) driven bioluminescence determinations using a luciferase-based assay system. Statistical correlations, linking ATP values to numbers of total bacteria, oral streptococci and mutans streptococci, yielded highly significant r values of 0.854, 0.840, and 0.796, respectively Our clinical data is consistent with the hypothesis that ATP measurements have a strong statistical association with bacterial number in plague and saliva specimens, including numbers for oral streptococci, and may be used as a potential assessment tool for oral hygiene and caries risk in children.

  8. A dual-function fluorescent probe for monitoring the degrees of hypoxia in living cells via the imaging of nitroreductase and adenosine triphosphate.

    PubMed

    Fang, Yu; Shi, Wen; Hu, Yiming; Li, Xiaohua; Ma, Huimin

    2018-05-24

    A new dual-function fluorescent probe is developed for detecting nitroreductase (NTR) and adenosine triphosphate (ATP) with different responses. Imaging application of the probe reveals that intracellular NTR and ATP display an adverse changing trend during a hypoxic process and ATP can serve as a new sign for cell hypoxia.

  9. Treatment of heterotopic ossification through remote ATP hydrolysis.

    PubMed

    Peterson, Jonathan R; De La Rosa, Sara; Eboda, Oluwatobi; Cilwa, Katherine E; Agarwal, Shailesh; Buchman, Steven R; Cederna, Paul S; Xi, Chuanwu; Morris, Michael D; Herndon, David N; Xiao, Wenzhong; Tompkins, Ronald G; Krebsbach, Paul H; Wang, Stewart C; Levi, Benjamin

    2014-09-24

    Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation. Copyright © 2014, American Association for the Advancement of Science.

  10. Effects of nucleotides adenosine monophosphate and adenosine triphosphate in combination with L-arginine on male rabbit corpus cavernosum tissue.

    PubMed

    Hupertan, V; Neuzillet, Y; Stücker, O; Pons, C; Leammel, E; Lebret, T

    2012-12-01

    Purines and more specifically adenosine monophosphate (AMP) and adenosine triphosphate (ATP) have a strong relaxant effect on smooth muscle cells of the dog, rabbit and human corpus cavernosum, to approximately the same degree as nitric oxide (NO). However, purines are considered as modulators of erectile function rather than key mediators. This suggests that the use of purines combined with NO donors could be effective to treat some specific erectile disorders. The relaxation induced by the combination of l-arginine (Arg), a natural substrate for NO synthase, was assessed with a purine-nucleotide (AMP, ATP) on a rabbit corpus cavernosum model, to determine if these substances could potentiate each other's effect. When a pre-contraction was induced by phenylephrine, AMP alone induced a 43% CC relaxation rate and ATP alone a 26% rate. The relaxation rate induced by Arg was lower in comparison (8% at 5.10(-4) m vs. 25% at AMP 5.10(-4) m and 15% at ATP 5.10(-4) m). NO synthase inhibitor n-nitro-l-arginine did not modify the relaxing effect provoked by AMP suggesting that the mechanism of action of this nucleotide does not involve the NO pathway. The combination of Arg at 5.10(-4) m with either AMP or ATP at different doses ranging from 5.10(-4) to 10(-3) m significantly enhanced the relaxing response reaching rates of 62 and 80% respectively, leading to a synergistic effect. The present data indicate that a 'NO donor' combined with an 'adenosine donor' could be an effective therapeutic approach. © 2012 The Authors. International Journal of Andrology © 2012 European Academy of Andrology.

  11. Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits.

    PubMed

    Kivi, Rait; Solovjova, Karina; Haljasorg, Tõiv; Arukuusk, Piret; Järv, Jaak

    2016-12-01

    The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

  12. A multifunctional probe based on the use of labeled aptamer and magnetic nanoparticles for fluorometric determination of adenosine 5'-triphosphate.

    PubMed

    Liu, Xiaojie; Lin, Bixia; Yu, Ying; Cao, Yujuan; Guo, Manli

    2018-04-02

    A multifunctional fluorescent probe is synthesized for the determination of adenosine 5'-triphosphate (ATP). The 6-carboxyfluorescein-labeled aptamer (FAM-aptamer) was bound to the surface of magnetite nanoparticles coated with polydopamine (Fe 3 O 4 @PDA) by π-π stacking interaction to form the multifunctional probe. The probe has three functions including recognition, magnetic separation, and yielding a fluorescent signal. In the presence of ATP, FAM-aptamer on the surface of the probe binds to ATP and returns to the solution. Thus, the fluorescence of the supernatant is enhanced and can be related to the concentration of ATP. Fluorescence intensities were measured at excitation/emission wavelengths of 494/526 nm. Response is linear in the 0.1-100 μM ATP concentration range, and the detection limit is 89 nM. The probe was applied to the quantitation of ATP in spiked human urine and serum samples, with recoveries ranging between 94.8 and 102%. Graphical abstract A multifunctional fluorescent probe based on the use of FAM-aptamer and Fe 3 O 4 @PDA is described for the determination of ATP in spiked human urine and serum samples. FAM-aptamer: 6-carboxyfluorescein-labeled aptamer; Fe 3 O 4 @PDA: magnetite nanoparticles coated with polydopamine. ATP: adenosine 5'-triphosphate.

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

    PubMed Central

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

    2014-01-01

    Organization of immune responses requires exchange of information between cells. This is achieved through either direct cell–cell contacts and establishment of temporary synapses or the release of soluble factors, such as cytokines and chemokines. Here we show a novel form of cell-to-cell communication based on adenosine triphosphate (ATP). ATP released by stimulated T cells induces P2X4/P2X7-mediated calcium waves in the neighboring lymphocytes. Our data obtained in lymph node slices suggest that, during T-cell priming, ATP acts as a paracrine messenger to reduce the motility of lymphocytes and that this may be relevant to allow optimal tissue scanning by T cells. PMID:24843045

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-20

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

  16. Adenosine 5'-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties.

    PubMed Central

    Guranowski, A; Starzyńska, E; Brown, P; Blackburn, G M

    1997-01-01

    Adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25+/-1 kDa. It catalyses the hydrolysis of a nucleoside 5'-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5'-tetraphosphate (p4A) is 8.2, Vmax is 21+/-1.7 micromol/min per mg of protein and the Km for p4A is 3+/-0.6 microM. At saturating p4A concentrations, the rate constant for the reaction is 8.5+/-0.7 s-1 [at 30 degrees C, in 50 mM Hepes/KOH (pH8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5'-tetraphosphate are hydrolysed at the same rate. Adenosine 5'-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively. PMID:9359862

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

    PubMed

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

    2015-10-01

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

  18. (-) Epicatechin attenuates mitochondrial damage by enhancing mitochondrial multi-marker enzymes, adenosine triphosphate and lowering calcium in isoproterenol induced myocardial infarcted rats.

    PubMed

    Stanely Mainzen Prince, P

    2013-03-01

    Cardiac mitochondrial damage plays an important role in the pathology of myocardial infarction. The protective effects of (-) epicatechin on cardiac mitochondrial damage in isoproterenol induced myocardial infarction were evaluated in rats. Rats were pretreated with (-) epicatechin (20 mg/kg body weight) daily for a period of 21 days. After the pretreatment period, isoproterenol (100 mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24 h to induce myocardial infarction. Isoproterenol induced myocardial infarcted rats showed a significant increase in the levels of cardiac diagnostic markers, heart mitochondrial lipid peroxidation, calcium, and a significant decrease in the activities/levels of heart mitochondrial glutathione peroxidase, glutathione reductase, reduced glutathione, isocitrate, succinate, malate, α-ketoglutarate and NADH-dehydrogenases, cytochrome-C-oxidase and adenosine triphosphate. (-) Epicatechin pretreatment showed significant protective effects on all the biochemical parameters evaluated. The in vitro study revealed the superoxide and hydroxyl radical scavenging activity of (-) epicatechin. The possible mechanisms for the beneficial effects of (-) epicatechin on cardiac mitochondria could be attributed to scavenging of free radicals, decreasing calcium, increasing multi-enzymes (antioxidant, tricarboxylic acid cycle and respiratory chain enzymes), reduced glutathione and adenosine triphosphate. Thus, (-) epicatechin attenuated mitochondrial damage in isoproterenol induced myocardial infarcted rats. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Intracellular Adenosine Triphosphate Delivery Enhanced Skin Wound Healing in Rabbits

    PubMed Central

    Wang, Jianpu; Zhang, Qunwei; Wan, Rong; Mo, Yiqun; Li, Ming; Tseng, Michael T.; Chien, Sufan

    2016-01-01

    Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery. This technique was tested in full-thickness skin wounds in 16 adult rabbits. One ear was rendered ischemic by using a minimally invasive surgery. The other ear served as a normal control. Four circular full-thickness wounds were created on the ventral side of each ear. ATP-vesicles or saline was used and the wounds were covered with Tegaderm (3M, St. Paul, MN). Dressing was changed and digital photos were taken daily until all the wounds were healed. The mean healing times of ATP-vesicles–treated wounds were significantly shorter than that of saline-treated wounds on ischemic and nonischemic ears. Histologic study indicated better-developed granular tissue and reepithelial-ization in the ATP-vesicles–treated wounds. The wounds treated by ATP-vesicles exhibited extremely fast granular tissue growth. More CD31 positive cells were seen in the ATP-vesicles–treated wounds. This preliminary study shows that direct intracellular delivery of ATP can accelerate the healing process of skin wounds on ischemic and nonischemic rabbit ears. The extremely fast granular tissue growth was something never seen or reported in the past. PMID:19158531

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

    PubMed

    Yukawa, Ayako; Watanabe, Rikiya; Noji, Hiroyuki

    2015-03-13

    F1-ATPase (F1), an important rotary motor protein, converts the chemical energy of ATP hydrolysis into mechanical energy using rotary motion with extremely high efficiency. The energy-conversion mechanism for this molecular motor has been extensively clarified by previous studies, which indicate that the interactions between the catalytic residues and the β- and γ-phosphates of ATP are indispensable for efficient catalysis and torque generation. However, the role of α-phosphate is largely unknown. In this study, we observed the rotation of F1 fuelled with an ATP analogue, adenosine 5'-[α-thio]-triphosphate (ATPαS), in which the oxygen has been substituted with a sulfur ion to perturb the α-phosphate/F1 interactions. In doing so, we have revealed that ATPαS does not appear to have any impact on the kinetic properties of the motor or on torque generation compared to ATP. On the other hand, F1 was observed to lapse into the ADP-inhibited intermediate states when in the presence of ATPαS more severely than in the presence of ATP, suggesting that the α-phosphate group of ATP contributes to the avoidance of ADP-inhibited intermediate formation. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-04-01

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

  2. Ultrasonic assisted synthesis of adenosine triphosphate capped manganese-doped ZnS quantum dots for selective room temperature phosphorescence detection of arginine and methylated arginine in urine based on supramolecular Mg(2+)-adenosine triphosphate-arginine ternary system.

    PubMed

    Ren, Hu-Bo; Yan, Xiu-Ping

    2012-08-15

    An ultrasonic assisted approach was developed for rapid synthesis of highly water soluble phosphorescent adenosine triphosphate (ATP)-capped Mn-doped ZnS QDs. The prepared ATP-capped Mn-doped ZnS QDs allow selective phosphorescent detection of arginine and methylated arginine based on the specific recognition nature of supramolecular Mg(2+)-ATP-arginine ternary system in combination with the phosphorescence property of Mn-doped ZnS QDs. The developed QD based probe gives excellent selectivity and reproducibility (1.7% relative standard deviation for 11 replicate detections of 10 μM arginine) and low detection limit (3 s, 0.23 μM), and favors biological applications due to the effective elimination of interference from scattering light and autofluorescence. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2015-01-01

    The hepatic cell death induced by acetaminophen (APAP) is closely related to cellular adenosine triphosphate (ATP) depletion, which is mainly caused by mitochondrial dysfunction. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a key sensor of low energy status. AMPK regulates metabolic homeostasis by stimulating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. We found that the decrease in active phosphorylation of AMPK in response to APAP correlates with decreased ATP levels, in vivo. Therefore, we hypothesized that the enhanced production of ATP via AMPK stimulation can lead to amelioration of APAP-induced liver failure. A769662, an allosteric activator of AMPK, produced a strong synergistic effect on AMPK Thr172 phosphorylation with APAP in primary hepatocytes and liver tissue. Interestingly, activation of AMPK by A769662 ameliorated the APAP-induced hepatotoxicity in C57BL/6N mice treated with APAP at a dose of 400 mg/kg intraperitoneally. However, mice treated with APAP alone developed massive centrilobular necrosis, and APAP increased their serum alanine aminotransferase and aspartate aminotransferase levels. Furthermore, A769662 administration prevented the loss of intracellular ATP without interfering with the APAP-mediated reduction of mitochondrial dysfunction. In contrast, inhibition of glycolysis by 2-deoxy-glucose eliminated the beneficial effects of A769662 on APAP-mediated liver injury. In conclusion, A769662 can effectively protect mice against APAP-induced liver injury through ATP synthesis by anaerobic glycolysis. Furthermore, stimulation of AMPK may have potential therapeutic application for APAP overdose. PMID:26434492

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

    PubMed

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

    2015-03-15

    Syncope is a significant source of cardiovascular-related morbidity yet the etiology is frequently obscure and the identification of patients at highest risk is challenging. Adenosine (AD) and adenosine triphosphate (ATP) administrations have been suggested as potentially useful non-invasive tools in the diagnostic workup of patients with neurally-mediated or bradycardia-related syncope. It has been postulated that both compounds by modulating the autonomic innervation in the heart and exerting negative chronotropic and dromotropic effects in the conduction system, may unmask the mechanism of syncope. However, the clinical implications derived from the efficacy of both tests in the investigation of syncope remain unclear mainly due to inconclusive and occasionally contradictory results of published studies. This review article summarizes recent and past information in the use of ATP and AD in the investigation of syncope with emphasis on clinical trials. We present the current level of evidence for the use of these agents in clinical practice, identify areas where further research is warranted and highlight the future perspectives of these agents as complements to an accurate risk-stratification of patients with syncope. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1981-10-01

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

  6. Simulating Protein Mediated Hydrolysis of ATP and Other Nucleoside Triphosphates by Combining QM/MM Molecular Dynamics with Advances in Metadynamics

    PubMed Central

    2017-01-01

    The protein mediated hydrolysis of nucleoside triphosphates such as ATP or GTP is one of the most important and challenging biochemical reactions in nature. The chemical environment (water structure, catalytic metal, and amino acid residues) adjacent to the hydrolysis site contains hundreds of atoms, usually greatly limiting the amount of the free energy sampling that one can achieve from computationally demanding electronic structure calculations such as QM/MM simulations. Therefore, the combination of QM/MM molecular dynamics with the recently developed transition-tempered metadynamics (TTMetaD), an enhanced sampling method that can provide a high-quality free energy estimate at an early stage in a simulation, is an ideal approach to address the biomolecular nucleoside triphosphate hydrolysis problem. In this work the ATP hydrolysis process in monomeric and filamentous actin is studied as an example application of the combined methodology. The performance of TTMetaD in these demanding QM/MM simulations is compared with that of the more conventional well-tempered metadynamics (WTMetaD). Our results show that TTMetaD exhibits much better exploration of the hydrolysis reaction free energy surface in two key collective variables (CVs) during the early stages of the QM/MM simulation than does WTMetaD. The TTMetaD simulations also reveal that a key third degree of freedom, the O–H bond-breaking and proton transfer from the lytic water, must be biased for TTMetaD to converge fully. To perturb the NTP hydrolysis dynamics to the least extent and to properly focus the MetaD free energy sampling, we also adopt here the recently developed metabasin metadynamics (MBMetaD) to construct a self-limiting bias potential that only applies to the lytic water after its nucleophilic attack of the phosphate of ATP. With these new, state-of-the-art enhanced sampling metadynamics techniques, we present an effective and accurate computational strategy for combining QM/MM molecular

  7. Simulating Protein Mediated Hydrolysis of ATP and Other Nucleoside Triphosphates by Combining QM/MM Molecular Dynamics with Advances in Metadynamics.

    PubMed

    Sun, Rui; Sode, Olaseni; Dama, James F; Voth, Gregory A

    2017-05-09

    The protein mediated hydrolysis of nucleoside triphosphates such as ATP or GTP is one of the most important and challenging biochemical reactions in nature. The chemical environment (water structure, catalytic metal, and amino acid residues) adjacent to the hydrolysis site contains hundreds of atoms, usually greatly limiting the amount of the free energy sampling that one can achieve from computationally demanding electronic structure calculations such as QM/MM simulations. Therefore, the combination of QM/MM molecular dynamics with the recently developed transition-tempered metadynamics (TTMetaD), an enhanced sampling method that can provide a high-quality free energy estimate at an early stage in a simulation, is an ideal approach to address the biomolecular nucleoside triphosphate hydrolysis problem. In this work the ATP hydrolysis process in monomeric and filamentous actin is studied as an example application of the combined methodology. The performance of TTMetaD in these demanding QM/MM simulations is compared with that of the more conventional well-tempered metadynamics (WTMetaD). Our results show that TTMetaD exhibits much better exploration of the hydrolysis reaction free energy surface in two key collective variables (CVs) during the early stages of the QM/MM simulation than does WTMetaD. The TTMetaD simulations also reveal that a key third degree of freedom, the O-H bond-breaking and proton transfer from the lytic water, must be biased for TTMetaD to converge fully. To perturb the NTP hydrolysis dynamics to the least extent and to properly focus the MetaD free energy sampling, we also adopt here the recently developed metabasin metadynamics (MBMetaD) to construct a self-limiting bias potential that only applies to the lytic water after its nucleophilic attack of the phosphate of ATP. With these new, state-of-the-art enhanced sampling metadynamics techniques, we present an effective and accurate computational strategy for combining QM/MM molecular

  8. Comparing visual inspection, aerobic colony counts, and adenosine triphosphate bioluminescence assay for evaluating surface cleanliness at a medical center.

    PubMed

    Huang, Yu-Shan; Chen, Yee-Chun; Chen, Mei-Ling; Cheng, Aristine; Hung, I-Chen; Wang, Jann-Tay; Sheng, Wang-Huei; Chang, Shan-Chwen

    2015-08-01

    Environmental cleaning is essential in reducing microbial colonization and health care-associated infections in hospitals. However, there is no consensus for the standard method to assess hospital cleanliness, and comparisons of newer methodology, such as adenosine triphosphate bioluminescence assay, with the traditional methods are limited. A prospective study was conducted at a medical center between January 2013 and August 2013. In each selected room, 10-12 high-touch surfaces were sampled before and after terminal cleaning. The adequacy of cleaning was evaluated by visual inspection, aerobic colony counts (ACCs), and adenosine triphosphate (ATP) bioluminescence assay. Eighty-five environmental surfaces from 8 rooms were evaluated by all 3 methods. The overall inadequacy defined by visual inspection, ACC, and ATP level was 11.8%, 20.0%, and 50.6% before cleaning and 4.7%, 5.9%, 21.2% after cleaning, respectively. A correlation between the ACC and ATP was found (r = 0.285, P < .001) using log10 values. Using ACCs <2.5 colony forming units/cm(2) as the cutoff for cleanliness, the ATP assay had better sensitivity than visual inspection (63.6% vs 27.3%). The receiver operating characteristics of the ATP assay indicated that the optimal ATP cutoff value was estimated to be 5.57 relative light units/cm(2). ATP bioluminescence assay is a sensitive and rapid tool in evaluating the quality of terminal cleaning. We emphasize the value of using a quantitative method to monitor environmental cleaning at hospitals. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-04-01

    Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1β, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1β mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1β from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury. © 2014 Wiley Publishing Asia Pty Ltd.

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

    PubMed

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

    2015-12-07

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

  11. Self-Assembled Tb3+ Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo.

    PubMed

    Jung, Sung Ho; Kim, Ka Young; Lee, Ji Ha; Moon, Cheol Joo; Han, Noh Soo; Park, Su-Jin; Kang, Dongmin; Song, Jae Kyu; Lee, Shim Sung; Choi, Myong Yong; Jaworski, Justyn; Jung, Jong Hwa

    2017-01-11

    To more accurately assess the pathways of biological systems, a probe is needed that may respond selectively to adenosine triphosphate (ATP) for both in vitro and in vivo detection modes. We have developed a luminescence probe that can provide real-time information on the extent of ATP, ADP, and AMP by virtue of the luminescence and luminescence lifetime observed from a supramolecular polymer based on a C 3 symmetrical terpyridine complex with Tb 3+ (S1-Tb). The probe shows remarkable selective luminescence enhancement in the presence of ATP compared to other phosphate-displaying nucleotides including adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), H 2 PO 4 - (Pi), and pyrophosphate (PPi). In addition, the time-resolved luminescence lifetime and luminescence spectrum of S1-Tb could facilitate the quantitative measurement of the exact amount of ATP and similarly ADP and AMP within living cells. The time-resolved luminescence lifetime of S1-Tb could also be used to quantitatively monitor the amount of ATP, ADP, and AMP in vitro following the enzymatic hydrolysis of ATP. The long luminescence lifetime, which was observed into the millisecond range, makes this S1-Tb-based probe particularly attractive for monitoring biological ATP levels in vivo, because any short lifetime background fluorescence arising from the complex molecular environment may be easily eliminated.

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

    NASA Technical Reports Server (NTRS)

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

    1977-01-01

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

  13. Adenosine Triphosphate-Encapsulated Liposomes with Plasmonic Nanoparticles for Surface Enhanced Raman Scattering-Based Immunoassays.

    PubMed

    Pham, Xuan-Hung; Hahm, Eunil; Kim, Tae Han; Kim, Hyung-Mo; Lee, Sang Hun; Lee, Yoon-Sik; Jeong, Dae Hong; Jun, Bong-Hyun

    2017-06-23

    In this study, we prepared adenosine triphosphate (ATP) encapsulated liposomes, and assessed their applicability for the surface enhanced Raman scattering (SERS)-based assays with gold-silver alloy (Au@Ag)-assembled silica nanoparticles (NPs; SiO₂@Au@Ag). The liposomes were prepared by the thin film hydration method from a mixture of l-α-phosphatidylcholine, cholesterol, and PE-PEG2000 in chloroform; evaporating the solvent, followed by hydration of the resulting thin film with ATP in phosphate-buffered saline (PBS). Upon lysis of the liposome, the SERS intensity of the SiO₂@Au@Ag NPs increased with the logarithm of number of ATP-encapsulated liposomes after lysis in the range of 8 × 10⁶ to 8 × 10 10 . The detection limit of liposome was calculated to be 1.3 × 10 -17 mol. The successful application of ATP-encapsulated liposomes to SiO₂@Au@Ag NPs based SERS analysis has opened a new avenue for Raman label chemical (RCL)-encapsulated liposome-enhanced SERS-based immunoassays.

  14. Aptamer-Based Carboxyl-Terminated Nanocrystalline Diamond Sensing Arrays for Adenosine Triphosphate Detection

    PubMed Central

    Suaebah, Evi; Naramura, Takuro; Myodo, Miho; Hasegawa, Masataka; Shoji, Shuichi; Buendia, Jorge J.; Kawarada, Hiroshi

    2017-01-01

    Here, we propose simple diamond functionalization by carboxyl termination for adenosine triphosphate (ATP) detection by an aptamer. The high-sensitivity label-free aptamer sensor for ATP detection was fabricated on nanocrystalline diamond (NCD). Carboxyl termination of the NCD surface by vacuum ultraviolet excimer laser and fluorine termination of the background region as a passivated layer were investigated by X-ray photoelectron spectroscopy. Single strand DNA (amide modification) was used as the supporting biomolecule to immobilize into the diamond surface via carboxyl termination and become a double strand with aptamer. ATP detection by aptamer was observed as a 66% fluorescence signal intensity decrease of the hybridization intensity signal. The sensor operation was also investigated by the field-effect characteristics. The shift of the drain current–drain voltage characteristics was used as the indicator for detection of ATP. From the field-effect characteristics, the shift of the drain current–drain voltage was observed in the negative direction. The negative charge direction shows that the aptamer is capable of detecting ATP. The ability of the sensor to detect ATP was investigated by fabricating a field-effect transistor on the modified NCD surface. PMID:28753998

  15. Aptamer-Based Carboxyl-Terminated Nanocrystalline Diamond Sensing Arrays for Adenosine Triphosphate Detection.

    PubMed

    Suaebah, Evi; Naramura, Takuro; Myodo, Miho; Hasegawa, Masataka; Shoji, Shuichi; Buendia, Jorge J; Kawarada, Hiroshi

    2017-07-21

    Here, we propose simple diamond functionalization by carboxyl termination for adenosine triphosphate (ATP) detection by an aptamer. The high-sensitivity label-free aptamer sensor for ATP detection was fabricated on nanocrystalline diamond (NCD). Carboxyl termination of the NCD surface by vacuum ultraviolet excimer laser and fluorine termination of the background region as a passivated layer were investigated by X-ray photoelectron spectroscopy. Single strand DNA (amide modification) was used as the supporting biomolecule to immobilize into the diamond surface via carboxyl termination and become a double strand with aptamer. ATP detection by aptamer was observed as a 66% fluorescence signal intensity decrease of the hybridization intensity signal. The sensor operation was also investigated by the field-effect characteristics. The shift of the drain current-drain voltage characteristics was used as the indicator for detection of ATP. From the field-effect characteristics, the shift of the drain current-drain voltage was observed in the negative direction. The negative charge direction shows that the aptamer is capable of detecting ATP. The ability of the sensor to detect ATP was investigated by fabricating a field-effect transistor on the modified NCD surface.

  16. Adenosine Triphosphate Promotes Allergen-Induced Airway Inflammation and Th17 Cell Polarization in Neutrophilic Asthma.

    PubMed

    Zhang, Fang; Su, Xin; Huang, Gang; Xin, Xiao-Feng; Cao, E-Hong; Shi, Yi; Song, Yong

    2017-01-01

    Adenosine triphosphate (ATP) is a key mediator to alert the immune dysfunction by acting on P2 receptors. Here, we found that allergen challenge caused an increase of ATP secretion in a murine model of neutrophilic asthma, which correlated well with neutrophil counts and interleukin-17 production. When ATP signaling was blocked by intratracheal administration of the ATP receptor antagonist suramin before challenge, neutrophilic airway inflammation, airway hyperresponsiveness, and Th17-type responses were reduced significantly. Also, neutrophilic inflammation was abrogated when airway ATP levels were locally neutralized using apyrase. Furthermore, ATP promoted the Th17 polarization of splenic CD4 + T cells from DO11.10 mice in vitro. In addition, ovalbumin (OVA) challenge induced neutrophilic inflammation and Th17 polarization in DO11.10 mice, whereas administration of suramin before challenge alleviated these parameters. Thus, ATP may serve as a marker of neutrophilic asthma, and local blockade of ATP signaling might provide an alternative method to prevent Th17-mediated airway inflammation in neutrophilic asthma.

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

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

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

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

    PubMed

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

    2018-04-01

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

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

    PubMed

    Chen, Lifen; Chen, Zhong-Ning

    2015-01-01

    A multifunctional label-free biosensor for the detection of Hg(2+), adenosine triphosphate and thrombin has been developed based on the changing of the electrochemical impedance spectroscopy (EIS) from the modified electrodes when nucleic acid subunits interacting with different targets. The modified electrode consists of three interaction sections, including DNA with T-T mismatch recognizing Hg(2+) to form T-Hg(2+)-T complex, split DNA chip against ATP, and DNA domin against thrombin to form G-quadruplex. Upon DNA interaction with thrombin or ATP, an increased charge transfer resistance (Rct) had been detected. However, a decreased Rct against Hg(2+) was obtained. The Rct difference (ΔRct) has relationship with the concentration of the different targets, Hg(2+), ATP and thrombin can be selectively detected with the detection limit of 0.03, 0.25, and 0.20 nmol L(-1), respectively. To separately detect the three analytes existing in the same sample, ATP aptamer, G-rich DNA strands and EDTA were applied to mask ATP, Hg(2+) or thrombin separately. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

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

    PubMed

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

    2016-05-01

    In the last years adenosine triphosphate (ATP) and subsequent purinergic system activation through P2 receptors were investigated highlighting their pivotal role in bone tissue biology. In osteoblasts ATP can regulate several activities like cell proliferation, cell death, cell differentiation and matrix mineralization. Since controversial results exist, in this study we analyzed the ATP effects on differentiation and mineralization in human osteoblast-like Saos-2 cells. We showed for the first time the altered functional activity of ATP receptors. Despite that, we found that ATP can reduce cell proliferation and stimulate osteogenic differentiation mainly in the early stages of in vitro maturation as evidenced by the enhanced expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and Osteocalcin (OC) genes and by the increased ALP activity. Moreover, we found that ATP can affect mineralization in a biphasic manner, at low concentrations ATP always increases mineral deposition while at high concentrations it always reduces mineral deposition. In conclusion, we show the osteogenic effect of ATP on both early and late stage activities like differentiation and mineralization, for the first time in human osteoblastic cells. © 2016 Japanese Society of Developmental Biologists.

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

    DOE PAGES

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

    2015-03-23

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

  3. Adenosine receptor activation potentiates phosphoinositide hydrolysis and arachidonic acid release in DDT1-MF2 cells: putative interrelations.

    PubMed

    Schachter, J B; Yasuda, R P; Wolfe, B B

    1995-09-01

    Studies were undertaken in an effort to discern possible mechanisms by which the A1 adenosine receptor agonist cyclopentyladenosine (CPA) enhances the norepinephrine-stimulated (NE-stimulated) hydrolysis of phosphoinositides in DDT1-MF2 cells. Measurements of arachidonic acid release revealed similar behaviours to those observed in measurements of phosphoinositide hydrolysis. In the presence of NE, both second messenger responses were potentiated by the addition of CPA, whereas in the absence of NE, CPA had little or no effect on either second messenger. The stimulation and potentiation of both second messenger responses were enhanced in the presence of extracellular calcium, and in each case these effects were persistent over time. For either second messenger system the stimulation by NE and the potentiation by CPA appeared to utilize separate mechanisms as evidenced by the fact that the potentiations by CPA were selectively antagonized by a cAMP analogue or by pertussis toxin, whereas the stimulations by NE were essentially unaffected by these agents. Inhibition of phospholipase A2 (PLA2) also blocked the potentiation of PLC by CPA, without affecting NE-stimulated phosphoinositide hydrolysis. Furthermore, in the presence of CPA, the exogenous administration of PLA2 was found to stimulate phosphoinositide hydrolysis in these cells. These data are consistent with a hypothesis whereby the apparent potentiation of NE-stimulated phosphoinositide hydrolysis by CPA is actually due to the stimulation by CPA of a second pathway of phospholipase C activity which is additive to that of NE. The activation of PLC and PLA2 by NE produces phospholipid products which may play a permissive role in the pathway coupling adenosine A1 receptors to these phospholipases. The formation of lysophosphatidic acid is suggested as one possible mediator of this permissive effect.

  4. Adenosine Triphosphate Quantification Correlates Poorly with Microbial Contamination of Duodenoscopes.

    PubMed

    Olafsdottir, Lovisa B; Wright, Sharon B; Smithey, Anne; Heroux, Riley; Hirsch, Elizabeth B; Chen, Alice; Lane, Benjamin; Sawhney, Mandeep S; Snyder, Graham M

    2017-06-01

    OBJECTIVE The aim of this study was to quantify the correlation between adenosine triphosphate (ATP) measurements and bacterial cultures from duodenoscopes for evaluation of contamination following high-level disinfection. DESIGN Duodenoscopes used for any intended endoscopic retrograde cholangiopancreatography (ERCP) procedure were included. Microbiologic and ATP data were collected concomitantly and in the same manner from ERCP duodenoscopes. SETTING A high-volume endoscopy unit at a tertiary referral acute-care facility. METHODS Duodenoscopes were sampled for ATP and bacterial contamination in a contemporaneous and highly standardized fashion using a "flush-brush-flush" method for the working channel (WC) and a dry flocked swab for the elevator mechanism (EM). Specimens were processed for any aerobic bacterial growth (colony-forming units, CFU). Growth of CFU>0 and ATP relative light unit (RLU)>0 was considered a contaminated result. Frequency of discord between among WC and EM measurements were calculated using 2×2 contingency tables. The Spearman correlation coefficient was used to calculate the relatedness of bacterial contamination and ATP as continuous measurements. RESULTS The Spearman correlation coefficient did not demonstrate significant relatedness between ATP and CFU for either a WC or EM site. Among 390 duodenoscope sampling events, ATP and CFU assessments of contamination were discordant in 82 of 390 WC measurements (21%) and 331 of 390 of EM measurements (84.9%). The EM was frequently and markedly positive by ATP measurement. CONCLUSION ATP measurements correlate poorly with a microbiologic standard assessing duodenoscope contamination, particularly for EM sampling. ATP may reflect biological material other than nonviable aerobic bacteria and may not serve as an adequate marker of bacterial contamination. Infect Control Hosp Epidemiol 2017;38:678-684.

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

    PubMed

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

    2016-03-01

    Adenosine triphosphate (ATP) is released onto the pinealocyte, along with noradrenaline, from sympathetic neurons and triggers P2Y1 receptors that enhance β-adrenergic-induced N-acetylserotonin (NAS) synthesis. Nevertheless, the biotransformation of NAS into melatonin, which occurs due to the subsequent methylation by acetylserotonin O-methyltransferase (ASMT; EC 2.1.1.4), has not yet been evaluated in the presence of purinergic stimulation. We therefore evaluated the effects of purinergic signaling on melatonin synthesis induced by β-adrenergic stimulation. ATP increased NAS levels, but, surprisingly, inhibited melatonin synthesis in an inverse, concentration-dependent manner. Our results demonstrate that enhanced NAS levels, which depend on phospholipase C (PLC) activity (but not the induction of gene transcription), are a post-translational effect. By contrast, melatonin reduction is related to an ASMT inhibition of expression at both the gene transcription and protein levels. These results were independent of nuclear factor-kappa B (NF-kB) translocation. Neither the P2Y1 receptor activation nor the PLC-mediated pathway was involved in the decrease in melatonin, indicating that ATP regulates pineal metabolism through different mechanisms. Taken together, our data demonstrate that purinergic signaling differentially modulates NAS and melatonin synthesis and point to a regulatory role for ATP as a cotransmitter in the control of ASMT, the rate-limiting enzyme in melatonin synthesis. The endogenous production of melatonin regulates defense responses; therefore, understanding the mechanisms involving ASMT regulation might provide novel insights into the development and progression of neurological disorders since melatonin presents anti-inflammatory, neuroprotective, and neurogenic effects. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed

    Long, Gong; Zhang, Guo Qiang

    2014-12-01

    Functional exercise after total knee arthroplasty (TKA) is necessary. However, it may be a difficult and painful process for the patient. Desirable methods of relieving the patient's pain are worth exploring. Oral supplement of adenosine triphosphate (ATP) is a potential option. In the present study, we decide to investigate whether short-term administration of ATP benefits patients undergoing TKA. A total of 244 subjects were randomized to receive 120mg ATP or placebo each day for 4weeks. Significant differences in quadriceps strength, pain scores at postoperative days 7, 14, 21, and 28 and total opioid consumption were detected. It follows that oral supplement of ATP could benefit patients recovering from TKA. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Electrochemical aptamer-based nanosensor fabricated on single Au nanowire electrodes for adenosine triphosphate assay.

    PubMed

    Wang, Dongmei; Xiao, Xiaoqing; Xu, Shen; Liu, Yong; Li, Yongxin

    2018-01-15

    In this work, single Au nanowire electrodes (AuNWEs) were fabricated by laser-assisted pulling/hydrofluoric acid (HF) etching process, which then were characterized by transmission electron microscopy (TEM), electrochemical method and finite-element simulation. The as-prepared single AuNWEs were used to construct electrochemical aptamer-based nanosensors (E-AB nanosensors) based on the formation of Au-S bond that duplex DNA tagged with methylene blue (MB) was modified on the surface of electrode. In the presence of adenosine triphosphate (ATP), the MB-labeled aptamer dissociated from the duplex DNA due to the strong specific affinity between aptamer and target, which lead to the reduction of MB electrochemical signals. Moreover, BSA was employed to further passivate electrode surface bonding sites for the stable of the sensor. The as-prepared E-AB nanosensor has been used for ATP assay with excellent sensitivity and selectivity, even in a complex system like cerebrospinal fluid of rat brain. Considering the unique properties of good stability, larger surface area and smaller overall dimensions, this E-AB nanosensor should be an ideal platform for widely sensing applications in living bio-system. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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

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

    PubMed

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

    2017-01-01

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

  10. Increased intracellular adenosine triphosphate level as an index to predict acute rejection in kidney transplant recipients.

    PubMed

    Wang, Xu-Zhen; Jin, Zhan-Kui; Tian, Xiao-Hui; Xue, Wu-Jun; Tian, Pu-Xun; Ding, Xiao-Ming; Zheng, Jin; Li, Yang; Jing, Xin; Luo, Zi-Zhen

    2014-01-01

    Peripheral blood CD4+ T cell adenosine triphosphate (ATP) release has been reported to be an adjunct tool to evaluate global cellular immune response in solid-organ transplant recipients. However, the correlation between the ATP level and rejection was controversial. The aim of this prospective clinical study was to explore the association between the intracellular ATP level and the occurrence, progression, and treatment of acute rejection (AR) episodes, determine the predicting value of intracellular ATP level for AR in kidney transplant (KT) recipients. In the period of October 2011 to October 2012, 140 KT recipients were recruited and followed for six months after transplantation. Patients were categorized into stable group and AR group according to their clinical course. Whole blood samples were collected pretransplantation, and at 7, 14, 21, and 28days, and at 2, 3, 4, 5 and 6months post-transplantation. Additional blood samples were obtained from AR patients on the day AR occurred, on the day before and 3 and 7days after intravenous anti-rejection therapy started, and on the day when AR reversed. The intracellular ATP in CD4+ T cells was detected by ImmuKnow Immune Cell Function Assay according to the manufacturer's instruction. The absolute number of CD4+ T cells and the trough levels of tacrolimus and cyclosporine were also measured. The ATP level detected on the day AR occurred (627.07±149.85ng/ml) was obviously higher than that of the stable group (320.48±149.11ng/ml, P<0.05). ATP value decreased to 265.35±84.33ng/m at the end of anti-rejection therapy, which was obviously lower than that measured on the day before the anti-rejection therapy started (665.87±162.85ng/ml, P<0.05). ROC analysis revealed that increased intracellular adenosine triphosphate level showed better sensitivity and specificity than those obtained using single time point detection (89.5% vs 85.0%;95.0% vs 88.9%). The best cutoff value was 172.55ng/ml. A positive correlation

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

    PubMed

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

    2015-09-07

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

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

    PubMed

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

    Adenosine triphosphate (ATP) analysis is a cultivation-independent alternative method for the determination of bacterial viability in both chlorinated and non-chlorinated water. Here we investigated the behavior and stability of ATP during chlorination in detail. Different sodium hypochlorite doses (0-22.4 mg-Cl2 L(-1); 5 min exposure) were applied to an Escherichia coli pure culture suspended in filtered river water. We observed decreasing intracellular ATP with increasing chlorine concentrations, but extracellular ATP concentrations only increased when the chlorine dose exceeded 0.35 mg L(-1). The release of ATP from chlorine-damaged bacteria coincided with severe membrane damage detected with flow cytometry (FCM). The stability of extracellular ATP was subsequently studied in different water matrixes, and we found that extracellular ATP was stable in sterile deionized water and also in chlorinated water until extremely high chlorine doses (≤11.2 mg-Cl2 L(-1); 5 min exposure). In contrast, ATP decreased relatively slowly (k = 0.145 h(-1)) in 0.1 μm filtered river water, presumably due to degradation by either extracellular enzymes or the fraction of bacteria that were able to pass through the filter. Extracellular ATP decreased considerably faster (k = 0.368 h(-1)) during batch growth of a river water bacterial community. A series of growth potential tests showed that extracellular ATP molecules were utilized as a phosphorus source during bacteria proliferation. From the combined data we conclude that ATP released from bacteria at high chlorine doses could promote bacteria regrowth, contributing to biological instability in drinking water distribution systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

    Villanueva, Ariadna; Guanche, Humberto

    2016-11-01

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

  14. Photoinduced Regeneration of an Aptamer-Based Electrochemical Sensor for Sensitively Detecting Adenosine Triphosphate.

    PubMed

    Zhang, Xiaoyu; Song, Chunxia; Yang, Ke; Hong, Wenwen; Lu, Ying; Yu, Ping; Mao, Lanqun

    2018-04-17

    Electrochemical aptasensors generally include three elements, that is, recognition element, signal-transformation element, and regeneration element. In this study, a new adenosine triphosphate (ATP) aptasensor is developed by combining three elements into one DNA oligonucleotide chain. In the DNA oligonucleotide chain, DNA aptamer is used as the recognition element, ferrocene group attached at the 3'-end of the aptamer is used as the signal-transformation element, and azobenzene moiety embedded into the DNA chain is used as the regeneration element. In addition to the similar analytical properties with the traditional ones, the aptasensor developed here is easily regenerated with UV-light irradiation. The current response recorded on the aptasensor increases with increasing the concentration of ATP in the incubation solution and is linear with the logarithm of ATP concentration in the range from 1 nM to 100 μM. The limit of detection is 0.5 nM (S/N = 3). The basal level of ATP in the rat brain cortex microdialysate is determined to be 21.33 ± 4.1 nM ( n = 3). After being challenged with ATP, the aptasensor could be readily regenerated by UV-light irradiation for more than seven cycles. The regeneration of the aptasensor is proposed to be regulated by conversing azobenzene from its trans to cis form under UV irradiation.

  15. Oral Adenosine-5'-triphosphate (ATP) Administration Increases Postexercise ATP Levels, Muscle Excitability, and Athletic Performance Following a Repeated Sprint Bout.

    PubMed

    Purpura, Martin; Rathmacher, John A; Sharp, Matthew H; Lowery, Ryan P; Shields, Kevin A; Partl, Jeremy M; Wilson, Jacob M; Jäger, Ralf

    2017-01-01

    Oral adenosine-5'-triphosphate (ATP) administration has failed to increase plasma ATP levels; however, chronic supplementation with ATP has shown to increase power, strength, lean body mass, and blood flow in trained athletes. The purpose of this study was to investigate the effects of ATP supplementation on postexercise ATP levels and on muscle activation and excitability and power following a repeated sprint bout. In a double-blind, placebo-controlled, randomized design, 42 healthy male individuals were given either 400 mg of ATP as disodium salt or placebo for 2 weeks prior to an exercise bout. During the exercise bout, muscle activation and excitability (ME, ratio of power output to muscle activation) and Wingate test peak power were measured during all sprints. ATP and metabolites were measured at baseline, after supplementation, and immediately following exercise. Oral ATP supplementation prevented a drop in ATP, adenosine-5'-diphosphate (ADP), and adenosine-5'-monophosphate (AMP) levels postexercise (p < 0.05). No group by time interaction was observed for muscle activation. Following the supplementation period, muscle excitability significantly decreased in later bouts 8, 9, and 10 in the placebo group (-30.5, -28.3, and -27.9%, respectively; p < 0.02), whereas ATP supplementation prevented the decline in later bouts. ATP significantly increased Wingate peak power in later bouts compared to baseline (bout 8: +18.3%, bout 10: +16.3%). Oral ATP administration prevents exercise-induced declines in ATP and its metabolite and enhances peak power and muscular excitability, which may be beneficial for sports requiring repeated high-intensity sprinting bouts.

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

    PubMed

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

    2013-08-01

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

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

    PubMed

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

    2014-03-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

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

  19. Facile synthesis of titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles for highly specific enrichment and analysis of intact phosphoproteins.

    PubMed

    Wang, Hao; Tian, Zhixin

    2018-06-06

    Analysis of phosphoproteins always faces the challenge of low stoichiometry, which demands highly selective and efficient enrichment in the initial sample preparation. Here we report our synthesis of the novel titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles (Ti 4+ -ATP-NPs) for efficient enrichment of intact phosphoproteins. The average diameter of Ti 4+ -ATP-NPs was about 128 nm with good dispersibility and the saturated adsorption capacity for β-casein was 1046.5 mg/g. In addition, Ti 4+ -ATP-NPs exhibited high specificity and selectivity in enriching phosphoproteins from both standard protein mixtures and complex biological samples (non-fat milk, chicken egg white and mouse heart tissue extract) as demonstrated by SDS-PAGE. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2014-06-03

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

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

    PubMed Central

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

    2015-01-01

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

  2. Adenosine and adenosine receptors in the pathogenesis and treatment of rheumatic diseases.

    PubMed

    Cronstein, Bruce N; Sitkovsky, Michail

    2017-01-01

    Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of inflammation. Adenosine mediates its effects on inflammatory cells by engaging one or more cell-surface receptors. The expression and function of adenosine receptors on different cell types change during the course of rheumatic diseases, such as rheumatoid arthritis (RA). Targeting adenosine receptors directly for the treatment of rheumatic diseases is currently under study; however, indirect targeting of adenosine receptors by enhancing adenosine levels at inflamed sites accounts for most of the anti-inflammatory effects of methotrexate, the anchor drug for the treatment of RA. In this Review, we discuss the regulation of extracellular adenosine levels and the role of adenosine in regulating the inflammatory and immune responses in rheumatic diseases such as RA, psoriasis and other types of inflammatory arthritis. In addition, adenosine and its receptors are involved in promoting fibrous matrix production in the skin and other organs, and the role of adenosine in fibrosis and fibrosing diseases is also discussed.

  3. Adenosine Triphosphate Regresses Endometrial Explants in a Rat Model of Endometriosis.

    PubMed

    Zhang, Chen; Gao, Li; Yi, Yanhong; Han, Hongjing; Cheng, Hongyan; Ye, Xue; Ma, Ruiqiong; Sun, Kunkun; Cui, Heng; Chang, Xiaohong

    2016-07-01

    The aim of this study was to determine the effects of adenosine triphosphate (ATP) in a rat endometriosis model. After surgical induction of endometriosis, 3 rats were killed, and explants were measured in the remaining 19 rats, which were then randomly assigned to 4 groups. Group 1 (n = 4) received normal saline (2 mL/d intragastric [IG]), group 2 (n = 4) gestrinone (0.5 mg/kg/d IG), group 3 (n = 5) ATP (3.4 mg/kg/d IG), and group 4 (n = 6) ATP (1.0 mg/kg/d; intramuscularly), respectively. Four weeks after medication, they were euthanized to evaluate histological features of explants and eutopic uterine tissues. To test the effect of ATP on the growth of eutopic endometrium stromal cells, proliferation rates of hEM15A cells at 24, 48, and 72 hours after treatment with different concentrations of ATP and vehicle control were detected with the Cell Counting Kit-8 (CCK-8) method. There was a significant difference between pretreatment and posttreatment volumes within group 2 (positive control; P = .048) and group 4 (P = .044). On condition that pretreatment implant size was similar in both groups (P = .516), regression of explants in group 4 was significantly higher than that in group 1 (negative control; P = .035). Epithelial cells were significantly better preserved in group 1 than in group 3 (P = .008) and group 4 (P = .037). The CCK-8 assay showed no significant difference in proliferation among hEM15A cells treated with ATP and controls. These results suggest that ATP regresses endometriotic tissues in a rat endometriosis model but has no impact on the growth of eutopic endometrium stromal cells. © The Author(s) 2016.

  4. Adenosine triphosphate-based chemotherapy response assay-guided chemotherapy in unresectable colorectal liver metastasis

    PubMed Central

    Hur, H; Kim, N K; Kim, H G; Min, B S; Lee, K Y; Shin, S J; Cheon, J H; Choi, S H

    2012-01-01

    Background: This study aims to evaluate the effectiveness of adenosine triphosphate-based chemotherapy response assay (ATP-CRA)-guided neoadjuvant chemotherapy for increasing resectability in patients with unresectable colorectal liver metastasis. Patients and methods: Patients were randomised into two groups: Group A was treated by conventional chemotherapy regimen and Group B was treated by chemotherapy regimen according to the ATP-CRA. Three chemotherapeutic agents (5-fluorouracil, oxaliplatin and irinotecan) were tested by ATP-CRA and more sensitive agents were selected. Either FOLFOX or FOLFIRI was administered. Between Group A and B, treatment response and resectability were compared. Results: Between November 2008 and October 2010, a total 63 patients were randomised to Group A (N=32) or Group B (N=31). FOLFOX was more preferred in Group A than in Group B (26 out of 32 (81.3%) vs 20 out of 31 (64.5%)). Group B showed better treatment response than Group A (48.4% vs 21.9%, P=0.027). The resectability of hepatic lesion was higher in Group B (35.5% vs 12.5%, P=0.032). Mean duration from chemotherapy onset to the time of liver resection was 11 cycles (range 4–12) in Group A and 8 cycles (range 8–16) in Group B. Conclusion: This study showed that tailored-chemotherapy based on ATP-CRA could improve the treatment response and resectability in initially unresectable colorectal liver metastasis. PMID:22068817

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

    PubMed

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

    2016-07-01

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

  6. Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

    PubMed

    Yamazaki, Yasuhiro; Yasui, Kenta; Hashizume, Takahiro; Suto, Arisa; Mori, Ayaka; Murata, Yuzuki; Yamaguchi, Masahiko; Ikari, Akira; Sugatani, Junko

    2015-10-01

    The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery. © 2015 by the American Association for the Study of Liver Diseases.

  7. Capture and quality control mechanisms for adenosine-5'-triphosphate binding.

    PubMed

    Li, Li; Martinis, Susan A; Luthey-Schulten, Zaida

    2013-04-24

    The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly casting mechanism that acts upon the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates.

  8. Cloning and bacterial expression of adenosine-5'-triphosphate sulfurylase from the enteric protozoan parasite Entamoeba histolytica.

    PubMed

    Nozaki, T; Arase, T; Shigeta, Y; Asai, T; Leustek, T; Takeuchi, T

    1998-12-08

    A gene encoding adenosine-5'-triphosphate sulfurylase (AS) was cloned from the enteric protozoan parasite Entamoeba histolytica by polymerase chain reaction using degenerate oligonucleotide primers corresponding to conserved regions of the protein from a variety of organisms. The deduced amino acid sequence of E. histolytica AS revealed a calculated molecular mass of 47925 Da and an unusual basic pI of 9.38. The amebic protein sequence showed 23-48% identities with AS from bacteria, yeasts, fungi, plants, and animals with the highest identities being to Synechocystis sp. and Bacillus subtilis (48 and 44%, respectively). Four conserved blocks including putative sulfate-binding and phosphate-binding regions were highly conserved in the E. histolytica AS. The upstream region of the AS gene contained three conserved elements reported for other E. histolytica genes. A recombinant E. histolytica AS revealed enzymatic activity, measured in both the forward and reverse directions. Expression of the E. histolytica AS complemented cysteine auxotrophy of the AS-deficient Escherichia coli strains. Genomic hybridization revealed that the AS gene exists as a single copy gene. In the literature, this is the first description of an AS gene in Protozoa.

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

    PubMed

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

    2014-02-01

    A strand displacement reaction (SDR) system that runs solely on oligonucleotides has been developed for the amplification detection of adenosine triphosphate (ATP). It involves a target-induced SDR and an entropy-driven catalytic cycle of two SDRs with five oligonucleotides, denoted as substrate, fuel, catalyst, C-1, and C-2. Catalyst, released from the ATP aptamer-catalyst duplex by ATP molecule, catalyzes the SDRs to finally form the substrate-fuel duplex. All of the intermediates in the catalytic SDR processes have been identified by polyacrylamide gel electrophoresis (PAGE) analysis. The introduction of ATP into the SDR system will induce the ATP aptamer to form G-quadruplex conformation so as to release catalyst and trigger the SDR cycle. When the substrate and C-2 oligonucleotides were labeled with a carboxyfluorescein (FAM) fluorophore and a 4-([4-(dimethylamino)phenyl]azo)benzoic acid (DABCYL) quencher, this SDR catalytic system exhibited a "turn-on" response for ATP. The condition for detecting ATP, such as Mg²⁺ concentration, has been optimized to afford a detection limit of 20 nM. This work provides an enzyme-free biosensing strategy and has potential application in aptamer-based biosensing. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. ATP Hydrolysis Mechanism in a Maltose Transporter Explored by QM/MM Metadynamics Simulation.

    PubMed

    Hsu, Wei-Lin; Furuta, Tadaomi; Sakurai, Minoru

    2016-11-03

    Translocation of substrates across the cell membrane by adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters depends on the energy provided by ATP hydrolysis within the nucleotide-binding domains (NBDs). However, the detailed mechanism remains unclear. In this study, we focused on maltose transporter NBDs (MalK 2 ) and performed a quantum mechanical/molecular mechanical (QM/MM) well-tempered metadynamics simulation to address this issue. We explored the free-energy profile along an assigned collective variable. As a result, it was determined that the activation free energy is approximately 10.5 kcal/mol, and the reaction released approximately 3.8 kcal/mol of free energy, indicating that the reaction of interest is a one-step exothermic reaction. The dissociation of the ATP γ-phosphate seems to be the rate-limiting step, which supports the so-called dissociative model. Moreover, Glu159, located in the Walker B motif, acts as a base to abstract the proton from the lytic water, but is not the catalytic base, which corresponds to an atypical general base catalysis model. We also observed two interesting proton transfers: transfer from the His192 ε-position nitrogen to the dissociated inorganic phosphate, Pi, and transfer from the Lys42 side chain to adenosine 5'-diphosphate β-phosphate. These proton transfers would stabilize the posthydrolysis state. Our study provides significant insight into the ATP hydrolysis mechanism in MalK 2 from a dynamical viewpoint, and this insight would be applicable to other ABC transporters.

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

    PubMed

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

    2017-02-01

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

  12. Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

    PubMed

    Tantry, Subramanyam J; Markad, Shankar D; Shinde, Vikas; Bhat, Jyothi; Balakrishnan, Gayathri; Gupta, Amit K; Ambady, Anisha; Raichurkar, Anandkumar; Kedari, Chaitanyakumar; Sharma, Sreevalli; Mudugal, Naina V; Narayan, Ashwini; Naveen Kumar, C N; Nanduri, Robert; Bharath, Sowmya; Reddy, Jitendar; Panduga, Vijender; Prabhakar, K R; Kandaswamy, Karthikeyan; Saralaya, Ramanatha; Kaur, Parvinder; Dinesh, Neela; Guptha, Supreeth; Rich, Kirsty; Murray, David; Plant, Helen; Preston, Marian; Ashton, Helen; Plant, Darren; Walsh, Jarrod; Alcock, Peter; Naylor, Kathryn; Collier, Matthew; Whiteaker, James; McLaughlin, Robert E; Mallya, Meenakshi; Panda, Manoranjan; Rudrapatna, Suresh; Ramachandran, Vasanthi; Shandil, Radha; Sambandamurthy, Vasan K; Mdluli, Khisi; Cooper, Christopher B; Rubin, Harvey; Yano, Takahiro; Iyer, Pravin; Narayanan, Shridhar; Kavanagh, Stefan; Mukherjee, Kakoli; Balasubramanian, V; Hosagrahara, Vinayak P; Solapure, Suresh; Ravishankar, Sudha; Hameed P, Shahul

    2017-02-23

    The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

  13. Elevated uric acid and adenosine triphosphate concentrations in bronchoalveolar lavage fluid of eosinophilic pneumonia.

    PubMed

    Kobayashi, Takehito; Nakagome, Kazuyuki; Noguchi, Toru; Kobayashi, Kiyoko; Ueda, Yutaka; Soma, Tomoyuki; Ikebuchi, Kenji; Nakamoto, Hidetomo; Nagata, Makoto

    2017-09-01

    Recent evidence has suggested that the innate immune response may play a role in the development of eosinophilic airway inflammation. We previously reported that uric acid (UA) and adenosine triphosphate (ATP), two important damage-associated molecular pattern molecules (DAMPs), activate eosinophil functions, suggesting that these molecules may be involved in the development of eosinophilic airway inflammation. The objective of this study was to measure the concentrations of DAMPs including UA and ATP in the bronchoalveolar lavage fluid (BALF) of patients with eosinophilic pneumonia (EP). BAL was performed in patients with EP including acute and chronic eosinophilic pneumonia, and in patients with hypersensitivity pneumonia, and sarcoidosis. UA, ATP, and cytokine concentrations in the BALF were then measured. The UA concentration was increased in the BALF of EP patients. UA concentrations correlated with eosinophil numbers, and with eosinophil-derived neurotoxin and interleukin (IL)-5 concentrations. Furthermore, the ATP concentration was increased in the BALF of EP patients and ATP concentrations correlated with UA concentrations. Moreover, IL-33 was increased in EP patients and IL-33 concentrations correlated with UA and ATP concentrations. The UA and ATP concentration was increased in the BALF of EP patients. UA concentrations correlated with eosinophil numbers, and with ATP and IL-33 concentrations. Our findings suggest that DAMPs such as UA and ATP play a role in the pathogenesis of EP. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  14. The Role of Magnesium for Geometry and Charge in GTP Hydrolysis, Revealed by Quantum Mechanics/Molecular Mechanics Simulations

    PubMed Central

    Rudack, Till; Xia, Fei; Schlitter, Jürgen; Kötting, Carsten; Gerwert, Klaus

    2012-01-01

    The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and Pi in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and Pi release back to the diphosphate. Furthermore, the Ras·Mg2+ complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger. PMID:22853907

  15. Validation of adenosine triphosphate to audit manual cleaning of flexible endoscope channels.

    PubMed

    Alfa, Michelle J; Fatima, Iram; Olson, Nancy

    2013-03-01

    Compliance with cleaning of flexible endoscope channels cannot be verified using visual inspection. Adenosine triphosphate (ATP) has been suggested as a possible rapid cleaning monitor for flexible endoscope channels. There have not been published validation studies to specify the level of ATP that indicates inadequate cleaning has been achieved. The objective of this study was to validate the Clean-Trace (3M Inc, St. Paul, MN) ATP water test method for monitoring manual cleaning of flexible endoscopes. This was a simulated use study using a duodenoscope as the test device. Artificial test soil containing 10(6) colony-forming units of Pseudomonas aeruginosa and Enterococcus faecalis was used to perfuse all channels. The flush sample method for the suction-biopsy (L1) or air-water channel (L2) using 40 and 20 mLs sterile reverse osmosis water, respectively, was validated. Residuals of ATP, protein, hemoglobin, and bioburden were quantitated from channel samples taken from uncleaned, partially cleaned, and fully cleaned duodenoscopes. The benchmarks for clean were as follows: <6.4 μg/cm(2) protein, <2.2 μg/cm(2) hemoglobin, and <4-log10 colony-forming units/cm(2) bioburden. The average ATP in clean channel samples was 27.7 RLUs and 154 RLUs for L1 and L2, respectively (<200 RLUs for all channels). The average protein, hemoglobin, and bioburden benchmarks were achieved if <200 RLUs were detected. If the channel sample was >200 RLUs, the residual organic and bioburden levels would exceed the acceptable benchmarks. Our data validated that flexible endoscopes that have complete manual cleaning will have <200 RLUs by the Clean-Trace ATP test. Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  16. Adenosine triphosphate as a molecular mediator of the vascular response to injury.

    PubMed

    Guth, Christy M; Luo, Weifung; Jolayemi, Olukemi; Chadalavada, Kalyan S; Komalavilas, Padmini; Cheung-Flynn, Joyce; Brophy, Colleen M

    2017-08-01

    Human saphenous veins used for arterial bypass undergo stretch injury at the time of harvest and preimplant preparation. Vascular injury promotes intimal hyperplasia, the leading cause of graft failure, but the molecular events leading to this response are largely unknown. This study investigated adenosine triphosphate (ATP) as a potential molecular mediator in the vascular response to stretch injury, and the downstream effects of the purinergic receptor, P2X7R, and p38 MAPK activation. A subfailure stretch rat aorta model was used to determine the effect of stretch injury on release of ATP and vasomotor responses. Stretch-injured tissues were treated with apyrase, the P2X7R antagonist, A438079, or the p38 MAPK inhibitor, SB203580, and subsequent contractile forces were measured using a muscle bath. An exogenous ATP (eATP) injury model was developed and the experiment repeated. Change in p38 MAPK phosphorylation after stretch and eATP tissue injury was determined using Western blotting. Noninjured tissue was incubated in the p38 MAPK activator, anisomycin, and subsequent contractile function and p38 MAPK phosphorylation were analyzed. Stretch injury was associated with release of ATP. Contractile function was decreased in tissue subjected to subfailure stretch, eATP, and anisomycin. Contractile function was restored by apyrase, P2X7R antagonism, and p38-MAPK inhibition. Stretch, eATP, and anisomycin-injured tissue demonstrated increased phosphorylation of p38 MAPK. Taken together, these data suggest that the vascular response to stretch injury is associated with release of ATP and activation of the P2X7R/P38 MAPK pathway, resulting in contractile dysfunction. Modulation of this pathway in vein grafts after harvest and before implantation may reduce the vascular response to injury. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-04-01

    A sensitive and label-free electrochemiluminescence (ECL) aptasensor for the detection of adenosine triphosphate (ATP) was successfully designed using host-guest recognition between a metallocyclodextrin complex, i.e., tris(bipyridine)ruthenium(II)-β-cyclodextrin [tris(bpyRu)-β-CD], and an ATP-binding aptamer. In the protocol, the NH2-terminated aptamer was immobilized on a glassy carbon electrode (GCE) by a coupling interaction. After host-guest recognition between tris(bpyRu)-β-CD and aptamer, the tris(bpyRu)-β-CD/aptamer/GCE produced a strong ECL signal as a result of the photoactive properties of tris(bpyRu)-β-CD. However, in the presence of ATP, the ATP/aptamer complex was formed preferentially, which restricted host-guest recognition, and therefore less tris(bpyRu)-β-CD was attached to the GCE surface, resulting in an obvious decrease in the ECL intensity. Under optimal determination conditions, an excellent logarithmic linear relationship between the ECL decrease and ATP concentration was obtained in the range 10.0-0.05 nM, with a detection limit of 0.01 nM at the S/N ratio of 3. The proposed ECL-based ATP aptasensor exhibited high sensitivity and selectivity, without time-consuming signal-labeling procedures, and is considered to be a promising model for detection of aptamer-specific targets. Copyright © 2014. Published by Elsevier B.V.

  18. Adenosine Triphosphate Metabolism Measured by Phosphorus Magnetic Resonance Spectroscopy: A Potential Biomarker for Multiple Sclerosis Severity.

    PubMed

    Kauv, Paul; Ayache, Samar S; Créange, Alain; Chalah, Moussa A; Lefaucheur, Jean-Pascal; Hodel, Jérôme; Brugières, Pierre

    2017-01-01

    Phosphorus magnetic resonance spectroscopy (31P-MRS) has previously shown abnormal changes in energy metabolites in the brain of multiple sclerosis (MS) patients. However, the relationship between these energy metabolites - particularly adenosine triphosphate (ATP) - and the disease severity remains unclear. The objective of this study was to determine whether measuring ATP metabolites can help to predict disease severity in MS patients. 31P-MRS at 3 tesla was performed in 9 relapsing remitting (RRMS), 9 secondary progressive MS patients (SPMS), and 10 age-matched healthy controls. ATP metabolites (expressed as %) in normally appearing white matter of the centrum semiovale were compared between patients and healthy controls. The relationship between Expanded Disability Status Scale (EDSS) and ATP metabolites was evaluated. RRMS and SPMS patients had higher phosphocreatine (PCr) and lower phosphodiesters than healthy controls. In addition, RRMS patients had higher β-ATP% than SPMS patients. β-ATP% was negatively correlated with EDSS in all patients. Our findings suggest a defective PCr metabolism in both patient groups, and a higher state of energy production in RRMS that might reflect a compensatory mechanism in face of the increased needs. The correlation of β-ATP with EDSS makes it a candidate biomarker for assessing MS disease severity. © 2017 S. Karger AG, Basel.

  19. Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wiley, J.S.; Dubyak, G.R.

    Extracellular adenosine triphosphate (ATP) is known to reversibly increase the cation permeability of a variety of freshly isolated and cultured cell types. In this study the effects of extracellular ATP were studied using peripheral blood lymphocytes (PBL) isolated from both normal subjects and from patients with chronic lymphocytic leukemia (CLL). Changes in the permeability to Na+, Rb+, and Li+ ions were measured using conventional isotope and flame photometry techniques. In addition, changes in cytosolic (Ca2+) were fluorimetrically monitored to assess possible changes in net Ca2+ influx. ATP produced a 12-fold increase in 22Na+ influx into CLL cells but only amore » 3.5-fold increase in this flux in PBL cells. A maximal response was produced by 0.1 mmol/L ATP in the absence of Mg2+, while a twofold molar excess of Mg2+ over ATP abolished the response. ATP had no effect on the passive (ouabain-insensitive) 86Rb+ influx into PBL cells but stimulated this flux by fivefold in the CLL cells. Li+ influx into CLL cells was also stimulated threefold by ATP. Under these same conditions ATP also produced a net increase in total cell Na and a decrease in total cell K in the CLL cells. Exclusion of two normally impermeable dyes, trypan blue and ethidium bromide, was not altered in the ATP-treated CLL cells. Finally, extracellular ATP (3 mmol/L) produced no significant change in the cytosolic (Ca2+) of normal, monocyte-depleted populations of PBL. Conversely, this same concentration of ATP produced a very rapid and a significant (an average threefold peak change) increase in the cytosolic (Ca2+) of cell preparations derived from five out of nine CLL patients. In these latter CLL cells, the ATP-induced elevation in cytosolic (Ca2+) appeared to be due to a net increase in Ca2+ influx, since no elevations were observed when the extracellular (Ca2+) was reduced to less than 0.1 mmol/L.« less

  20. A Method to Determine 18O Kinetic Isotope Effects in the Hydrolysis of Nucleotide Triphosphates

    PubMed Central

    Du, Xinlin; Ferguson, Kurt; Sprang, Stephen R.

    2007-01-01

    A method to determine 18O kinetic isotope effects (KIE) in the hydrolysis of GTP is described that is generally applicable to reactions involving other nucleotide triphosphates. Internal competition, wherein the substrate of the reaction is a mixture of 18O-labeled and unlabeled nucleotides, is employed and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18O at sites of mechanistic interest also contains 13C at all carbon positions, while the 16O-nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink™ interface (ThermoFinnigan). Carbon isotope ratios can be determined with accuracy and precision greater than 0.04%, and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1333-catalyzed hydrolysis of [β18O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (< 0.1%). A single KIE measurement can be conducted in 25 minutes with less than 5 μg nucleotide reaction product. PMID:17963711

  1. Standard Gibbs energy of metabolic reactions: II. Glucose-6-phosphatase reaction and ATP hydrolysis.

    PubMed

    Meurer, Florian; Do, Hoang Tam; Sadowski, Gabriele; Held, Christoph

    2017-04-01

    ATP (adenosine triphosphate) is a key reaction for metabolism. Tools from systems biology require standard reaction data in order to predict metabolic pathways accurately. However, literature values for standard Gibbs energy of ATP hydrolysis are highly uncertain and differ strongly from each other. Further, such data usually neglect the activity coefficients of reacting agents, and published data like this is apparent (condition-dependent) data instead of activity-based standard data. In this work a consistent value for the standard Gibbs energy of ATP hydrolysis was determined. The activity coefficients of reacting agents were modeled with electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT). The Gibbs energy of ATP hydrolysis was calculated by combining the standard Gibbs energies of hexokinase reaction and of glucose-6-phosphate hydrolysis. While the standard Gibbs energy of hexokinase reaction was taken from previous work, standard Gibbs energy of glucose-6-phosphate hydrolysis reaction was determined in this work. For this purpose, reaction equilibrium molalities of reacting agents were measured at pH7 and pH8 at 298.15K at varying initial reacting agent molalities. The corresponding activity coefficients at experimental equilibrium molalities were predicted with ePC-SAFT yielding the Gibbs energy of glucose-6-phosphate hydrolysis of -13.72±0.75kJ·mol -1 . Combined with the value for hexokinase, the standard Gibbs energy of ATP hydrolysis was finally found to be -31.55±1.27kJ·mol -1 . For both, ATP hydrolysis and glucose-6-phosphate hydrolysis, a good agreement with own and literature values were obtained when influences of pH, temperature, and activity coefficients were explicitly taken into account in order to calculate standard Gibbs energy at pH7, 298.15K and standard state. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Adenosine production by human B cells and B cell–mediated suppression of activated T cells

    PubMed Central

    Saze, Zenichiro; Schuler, Patrick J.; Hong, Chang-Sook; Cheng, Dongmei; Jackson, Edwin K.

    2013-01-01

    Antibody-independent role of B cells in modulating T-cell responses is incompletely understood. Freshly isolated or cultured B cells isolated from the peripheral blood of 30 normal donors were evaluated for CD39 and CD73 coexpression, the ability to produce adenosine 5′-monophosphate (AMP) and adenosine (ADO) in the presence of exogenous adenosine triphosphate (ATP) as well as A1, A2A, A2B, and A3 adenosine receptor (ADOR) expression. Human circulating B cells coexpress ectonucleotidases CD39 and CD73, hydrolyze exogenous ATP to 5′-AMP and ADO, and express messenger RNA for A1R, A2AR, and A3R. 2-chloroadenosine inhibited B-cell proliferation and cytokine expression, and only A3R selective antagonist restored B-cell functions. This suggested that B cells use the A3R for autocrine signaling and self-regulation. Mediated effects on B-cell growth ± ADOR antagonists or agonists were tested in carboxyfluorescein diacetate succinimidyl ester assays. In cocultures, resting B cells upregulated functions of CD4+ and CD8+ T cells. However, in vitro–activated B cells downregulated CD73 expression, mainly produced 5′-AMP, and inhibited T-cell proliferation and cytokine production. These B cells acquire the ability to restrict potentially harmful effects of activated T cells. Thus, B cells emerge as a key regulatory component of T cell–B cell interactions, and their dual regulatory activity is mediated by the products of ATP hydrolysis, 5′-AMP, and ADO. PMID:23678003

  3. [High performance liquid chromatogram (HPLC) determination of adenosine phosphates in rat myocardium].

    PubMed

    Miao, Yu; Wang, Cheng-long; Yin, Hui-jun; Shi, Da-zhuo; Chen, Ke-ji

    2005-04-18

    To establish method for the quantitative determination of adenosine phosphates in rat myocardium by optimized high performance liquid chromatogram (HPLC). ODS HYPERSIL C(18) column and a mobile phase of 50 mmol/L tribasic potassium phosphate buffer solution (pH 6.5), with UV detector at 254 nm were used. The average recovery rates of myocardial adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were 99%-107%, 96%-104% and 95%-119%, respectively; relative standard deviations (RSDs) of within-day and between-days were less than 1.5% and 5.1%, respectively. The method is simple, rapid and accurate, and can be used to analyse the adenosine phosphates in myocardium.

  4. Cultured astrocytes do not release adenosine during hypoxic conditions

    PubMed Central

    Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

    2012-01-01

    Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia. PMID:21989480

  5. A quantitative analysis of the effects of 2,3-diphosphoglycerate, adenosine triphosphate and inositol hexaphosphate on the oxygen dissociation curve of human haemoglobin.

    PubMed Central

    Goodford, P J; St-Louis, J; Wootton, R

    1978-01-01

    1. Oxygen dissociation curves have been measured for human haemoglobin solutions with different concentrations of the allosteric effectors 2,3-diphosphoglycerate, adenosine triphosphate and inositol hexaphosphate. 2. Each effector produces a concentration dependent right shift of the oxygen dissociation curve, but a point is reached where the shift is maximal and increasing the effector concentration has no further effect. 3. Mathematical models based on the Monod, Wyman & Changeux (1965) treatment of allosteric proteins have been fitted to the data. For each compound the simple two-state model and its extension to take account of subunit inequivalence were shown to be inadequate, and a better fit was obtained by allowing the effector to lower the oxygen affinity of the deoxy conformational state as well as binding preferentially to this conformation. PMID:722582

  6. Prostasomes from four different species are able to produce extracellular adenosine triphosphate (ATP).

    PubMed

    Ronquist, K Göran; Ek, Bo; Morrell, Jane; Stavreus-Evers, Anneli; Ström Holst, Bodil; Humblot, Patrice; Ronquist, Gunnar; Larsson, Anders

    2013-10-01

    Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called "storage vesicle" equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization. Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species. The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10-150kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes. These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum. This study unravels energy metabolic relationships of prostasomes from four different species. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Use of adenosine triphosphate to audit reprocessing of flexible endoscopes with an elevator mechanism.

    PubMed

    Quan, Erik; Mahmood, Rizwan; Naik, Amar; Sargon, Peter; Shastri, Nikhil; Venu, Mukund; Parada, Jorge P; Gupta, Neil

    2018-05-21

    There have been reported outbreaks of carbapenem-resistant Enterobacteriaceae infections linked to endoscopes with elevator mechanisms. Adenosine triphosphate (ATP) testing has been used as a marker for bioburden and monitoring manual cleaning for flexible endoscopes with and without an elevator mechanism. The objective of this study was to determine whether routine ATP testing could identify areas of improvement in cleaning of endoscopes with an elevator mechanism. ATP testing after manual cleaning of TJF-Q180V duodenoscopes and GF-UCT180 linear echoendoscopes (Olympus America Inc, Center Valley, PA) was implemented. Samples were tested from the distal end, the elevator mechanism, and water flushed through the lumen of the biopsy channel. Data were recorded and compared by time point, test point, and reprocessing technician. Overall failure rate was 6.99% (295 out of 4,219). The highest percentage of failed ATP tests (17.05%) was reported in the first quarter of routine testing, with an overall decrease in rates over time. The elevator mechanism and working channel lumen had higher failure rates than the distal end. Quality of manual cleaning between reprocessing technicians showed variation. ATP testing is effective in identifying residual organic material and improving quality of manual cleaning of endoscopes with an elevator mechanism. Cleaning efficacy is influenced by reprocessing technicians and location tested on the endoscope. Close attention to the working channel and elevator mechanism during manual cleaning is warranted. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  8. Measurement of adenosine triphosphate and 2,3-diphosphoglycerate in stored blood with 31P nuclear magnetic resonance spectroscopy.

    PubMed

    Ambruso, D R; Hawkins, B; Johnson, D L; Fritzberg, A R; Klingensmith, W C; McCabe, E R

    1986-06-01

    Conditions for blood storage are chosen to assure adequate levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG). Because of the invasive nature of the techniques, biochemical assays are not routinely used to measure levels of these compounds in stored blood. However, 31P NMR spectroscopy measures phosphorylated intermediates in intact cells and could be used without disruption of the storage pack. We compared levels of ATP and 2,3-DPG measured by 31P spectroscopy and standard enzyme-linked biochemical assays in whole blood (WB) and packed red blood cells (PRBCs) at weekly intervals during a 35-day storage period. NMR demonstrated a marked decrease in 2,3-DPG and an increase in inorganic phosphate after the first week of storage. No significant differences in ATP concentrations were seen in WB during the storage period, but a significant decrease in ATP in PRBCs was documented. There was good agreement in levels of ATP and 2,3-DPG measured by NMR and biochemical techniques. 31P NMR spectroscopy is a noninvasive technique for measuring ATP and 2,3-DPG which has a potential use in quality assurance of stored blood.

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

    USGS Publications Warehouse

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

    2009-01-01

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

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

    PubMed Central

    Wagner, Marc C.E.

    2011-01-01

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

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

    PubMed

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

    2013-11-13

    In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8×10(-12) M to 2.40×10(-4) M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  12. Immune function monitoring in lung transplantation using adenosine triphosphate production: time trends and relationship to postoperative infection.

    PubMed

    Takahashi, Mamoru; Ohsumi, Akihiro; Ohata, Keiji; Kondo, Takeshi; Motoyama, Hideki; Hijiya, Kyoko; Aoyama, Akihiro; Date, Hiroshi; Chen-Yoshikawa, Toyofumi F

    2017-06-01

    The ImmuKnow (IK) assay is a comprehensive immune function test that involves measuring adenosine triphosphate produced by the cluster of differentiation 4+ T lymphocytes in peripheral blood. The aim of this study was to analyze the time trends of IK values and assess the relationship between IK values and infections in lung transplants. We prospectively collected 178 blood samples from 22 deceased-donor lung transplant (DDLT) recipients and 17 living-donor lobar lung transplant (LDLLT) recipients. A surveillance IK assay was performed postoperatively, then after 1 week and 1, 3, 6, and 12 months. Time trends of IK values in stable recipients peaked 1 week after DDLT (477 ± 247 ATP ng/ml), and 1 month after LDLLT (433 ± 134 ng/ml), followed by a gradual decline over 1 year. The mean IK values in infections were significantly lower than those in the stable state (119 vs 312 ATP ng/ml, p = 0.0002). IK values increased sharply after lung transplantation and then decreased gradually over time in the first year, suggesting a natural history of immune function. IK values were also significantly reduced during infections. These results may provide new insights into the utility of immune monitoring after lung transplantation.

  13. Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

    PubMed

    Wu, Liping; Oshima, Tadayuki; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2017-07-01

    Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

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

    PubMed Central

    Mittelstädt, Gerd; Moggré, Gert‐Jan; Panjikar, Santosh; Nazmi, Ali Reza

    2016-01-01

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

  15. Antifouling aptasensor for the detection of adenosine triphosphate in biological media based on mixed self-assembled aptamer and zwitterionic peptide.

    PubMed

    Wang, Guixiang; Su, Xiaoli; Xu, Qingjun; Xu, Guiyun; Lin, Jiehua; Luo, Xiliang

    2018-03-15

    Direct detection of targets in complex biological media with conventional biosensors is an enormous challenge due to the nonspecific adsorption and severe biofouling. In this work, a facile strategy for sensitive and low fouling detection of adenosine triphosphate (ATP) is developed through the construction of a mixed self-assembled biosensing interface, which was composed of zwitterionic peptide (antifouling material) and ATP aptamer (bio-recognition element). The peptide and aptamer (both containing thiol groups) were simultaneously self-assembled onto gold electrode surface electrodeposited with gold nanoparticles. The developed aptasensor possessed high selectivity and sensitivity for ATP, and it showed a wide linear response range towards ATP from 0.1pM to 5nM. Owing to the presence of peptide with excellent antifouling property in the biosensing interface, the aptasensor can detect ATP in complex biological media with remarkably reduced biofouling or nonspecific adsorption effect. Moreover, it can directly detect ATP in 1% human whole blood without suffering from any significant interference, indicating its great potential for practical assaying of ATP in biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-08-01

    Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. © 2016 The Protein Society.

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

    PubMed

    Ratajczak, Katarzyna; Stobiecka, Magdalena

    2017-07-20

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

  18. Duodenoscope reprocessing surveillance with adenosine triphosphate testing and terminal cultures: a clinical pilot study.

    PubMed

    Visrodia, Kavel; Hanada, Yuri; Pennington, Kelly M; Tosh, Pritish K; Topazian, Mark D; Petersen, Bret T

    2017-07-01

    Recent reports of infectious outbreaks linked to duodenoscopes have led to proposals for duodenoscope surveillance culturing, which has inherent limitations. We aimed to assess the feasibility of real-time adenosine triphosphate (ATP) testing after manual cleaning and its ability to predict reprocessing adequacy, as determined by terminal duodenoscope cultures. Clinically used duodenoscopes underwent reprocessing per current guidelines. After manual cleaning, ATP samples were obtained from the elevator, within the proximal biopsy port, and by flushing of the biopsy channel. After high-level disinfection (HLD), aerobic cultures of the elevator and biopsy channel were obtained using sterile technique. Duodenoscopes with any ATP sample ≥200 relative light units underwent repeated cycles of cleaning, ATP testing, HLD, and terminal culturing. Twenty clinically used duodenoscopes were included; 18 underwent a second reprocessing cycle, and 6 underwent a third reprocessing cycle because of detection of high ATP. After the initial reprocessing cycle, 12 of 20 (60%) duodenoscopes had positive culture results, most commonly yielding gram-negative bacilli (GNB, n = 11 from 9 duodenoscopes), and catalase-positive gram-positive cocci (CP-GPC, n = 7 from 7 duodenoscopes), suggesting staphylococcal organisms. Ambient environmental controls also showed GNB and CP-GPC growth. The overall sensitivity and specificity of ATP testing compared with terminal cultures were 30% and 53%, respectively. ATP sampling appears to correlate poorly with terminal culture results and cannot be recommended as a surrogate for terminal cultures. The performance and interpretation of cultures remains complicated by the potential recovery of environmental contaminants. Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

  19. Visual and surface plasmon resonance sensor for zirconium based on zirconium-induced aggregation of adenosine triphosphate-stabilized gold nanoparticles.

    PubMed

    Qi, Wenjing; Zhao, Jianming; Zhang, Wei; Liu, Zhongyuan; Xu, Min; Anjum, Saima; Majeed, Saadat; Xu, Guobao

    2013-07-17

    Owing to its high affinity with phosphate, Zr(IV) can induce the aggregation of adenosine 5'-triphosphate (ATP)-stabilized AuNPs, leading to the change of surface plasmon resonance (SPR) absorption spectra and color of ATP-stabilized AuNP solutions. Based on these phenomena, visual and SPR sensors for Zr(IV) have been developed for the first time. The A(660 nm)/A(518 nm) values of ATP-stabilized AuNPs in SPR absorption spectra increase linearly with the concentrations of Zr(IV) from 0.5 μM to 100 μM (r=0.9971) with a detection limit of 95 nM. A visual Zr(IV) detection is achieved with a detection limit of 30 μM. The sensor shows excellent selectivity against other metal ions, such as Cu(2+), Fe(3+), Cd(2+), and Pb(2+). The recoveries for the detection of 5 μM, 10 μM, 25 μM and 75 μM Zr(IV) in lake water samples are 96.0%, 97.0%, 95.6% and 102.4%, respectively. The recoveries of the proposed SPR method are comparable with those of ICP-OES method. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

    PubMed

    Kutryb-Zajac, Barbara; Mateuszuk, Lukasz; Zukowska, Paulina; Jasztal, Agnieszka; Zabielska, Magdalena A; Toczek, Marta; Jablonska, Patrycja; Zakrzewska, Agnieszka; Sitek, Barbara; Rogowski, Jan; Lango, Romuald; Slominska, Ewa M; Chlopicki, Stefan; Smolenski, Ryszard T

    2016-11-01

    Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular

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

    PubMed

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

    2016-11-15

    The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Inhibition of Dengue Virus RNA Synthesis by an Adenosine Nucleoside ▿ †

    PubMed Central

    Chen, Yen-Liang; Yin, Zheng; Duraiswamy, Jeyaraj; Schul, Wouter; Lim, Chin Chin; Liu, Boping; Xu, Hao Ying; Qing, Min; Yip, Andy; Wang, Gang; Chan, Wai Ling; Tan, Hui Pen; Lo, Melissa; Liung, Sarah; Kondreddi, Ravinder Reddy; Rao, Ranga; Gu, Helen; He, Handan; Keller, Thomas H.; Shi, Pei-Yong

    2010-01-01

    We recently reported that (2R,3R,4R,5R)-2-(4-amino-pyrrolo[2,3-d]pyrimidin-7-yl)-3-ethynyl-5-hydroxy-methyl-tetrahydro-furan-3,4-diol is a potent inhibitor of dengue virus (DENV), with 50% effective concentration (EC50) and cytotoxic concentration (CC50) values of 0.7 μM and >100 μM, respectively. Here we describe the synthesis, structure-activity relationship, and antiviral characterization of the inhibitor. In an AG129 mouse model, a single-dose treatment of DENV-infected mice with the compound suppressed peak viremia and completely prevented death. Mode-of-action analysis using a DENV replicon indicated that the compound blocks viral RNA synthesis. Recombinant adenosine kinase could convert the compound to a monophosphate form. Suppression of host adenosine kinase, using a specific inhibitor (iodotubercidin) or small interfering RNA (siRNA), abolished or reduced the compound's antiviral activity in cell culture. Studies of rats showed that 14C-labeled compound was converted to mono-, di-, and triphosphate metabolites in vivo. Collectively, the results suggest that this adenosine inhibitor is phosphorylated to an active (triphosphate) form which functions as a chain terminator for viral RNA synthesis. PMID:20457821

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

    USGS Publications Warehouse

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

    2009-01-01

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

  4. An easy and fast adenosine 5'-diphosphate quantification procedure based on hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry for determination of the in vitro adenosine 5'-triphosphatase activity of the human breast cancer resistance protein ABCG2.

    PubMed

    Wagmann, Lea; Maurer, Hans H; Meyer, Markus R

    2017-10-27

    Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5'-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5'-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5'-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Purinergic signaling pathways in endocrine system.

    PubMed

    Bjelobaba, Ivana; Janjic, Marija M; Stojilkovic, Stanko S

    2015-09-01

    Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. Published by Elsevier B.V.

  6. Purinergic Signaling Pathways in Endocrine System

    PubMed Central

    Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

    2015-01-01

    Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. PMID:25960051

  7. Increased NTPDase Activity in Lymphocytes during Experimental Sepsis

    PubMed Central

    Bertoncheli, Claudia de Mello; Zimmermann, Carine Eloise Prestes; Jaques, Jeandre Augusto dos Santos; Leal, Cláudio Alberto Martins; Ruchel, Jader Betsch; Rocha, Bruna Cipolatto; Pinheiro, Kelly de Vargas; Souza, Viviane do Carmo Gonçalves; Stainki, Daniel Roulim; Luz, Sônia Cristina Almeida; Schetinger, Maria Rosa Chitolina; Leal, Daniela Bitencourt Rosa

    2012-01-01

    We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C. 3.6.1.5), an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5′-triphosphate (ATP) (P < 0.01), but no changes regarding adenosine-5′-monophosphate (ADP) hydrolysis (P > 0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death. PMID:22645477

  8. 7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7IdTP incorporation during PCR-amplification and protection from endodeoxyribonuclease hydrolysis.

    PubMed Central

    Seela, F; Röling, A

    1992-01-01

    The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c7Gd), -adenosine (c7Ad) and -inosine (c7Id). c7GdTP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c7AdTP) and (c7IdTP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c7GdTP much better than c7AdTP or c7IdTP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phosphodiester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c7Gd is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae III, Rsa I, Hind III, Pvu II or Taq I) do still hydrolyze the modified DNA. c7Ad protects DNA less efficiently, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis. Images PMID:1738604

  9. Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation: coordination by extracellular nucleotide metabolism.

    PubMed

    Eltzschig, Holger K; Thompson, Linda F; Karhausen, Jorn; Cotta, Richard J; Ibla, Juan C; Robson, Simon C; Colgan, Sean P

    2004-12-15

    Hypoxia is a well-documented inflammatory stimulus and results in tissue polymorphonuclear leukocyte (PMN) accumulation. Likewise, increased tissue adenosine levels are commonly associated with hypoxia, and given the anti-inflammatory properties of adenosine, we hypothesized that adenosine production via adenine nucleotide metabolism at the vascular surface triggers an endogenous anti-inflammatory response during hypoxia. Initial in vitro studies indicated that endogenously generated adenosine, through activation of PMN adenosine A(2A) and A(2B) receptors, functions as an antiadhesive signal for PMN binding to microvascular endothelia. Intravascular nucleotides released by inflammatory cells undergo phosphohydrolysis via hypoxia-induced CD39 ectoapyrase (CD39 converts adenosine triphosphate/adenosine diphosphate [ATP/ADP] to adenosine monophosphate [AMP]) and CD73 ecto-5'-nucleotidase (CD73 converts AMP to adenosine). Extensions of our in vitro findings using cd39- and cd73-null animals revealed that extracellular adenosine produced through adenine nucleotide metabolism during hypoxia is a potent anti-inflammatory signal for PMNs in vivo. These findings identify CD39 and CD73 as critical control points for endogenous adenosine generation and implicate this pathway as an innate mechanism to attenuate excessive tissue PMN accumulation.

  10. A comparison of the chronotropic and dromotropic actions between adenosine triphosphate and edrophonium in patients undergoing coronary artery bypass graft surgery.

    PubMed

    Watanabe, Seiji; Kono, Yasuo; Oishi-Tobinaga, Yoko; Yamada, Shin-ichi; Hara, Masato; Kano, Tatsuhiko

    2002-10-01

    To compare the effects of the stimulation of adenosine receptors and acetylcholine receptors in the cardiac conduction system in patients with ischemic heart disease. Prospective. University hospital. Patients scheduled for coronary artery bypass graft surgery (n = 37). The patients were divided into 3 groups: control group (n = 9), adenosine triphosphate (ATP) group (n = 12), and edrophonium group (n = 16). ATP (10 mg) or edrophonium (0.25 mg/kg) followed by saline or the same amount of saline was injected through a central venous catheter. ATP induced atrioventricular block in 10 of 12 patients (83%). The ATP injection produced a more prominent prolongation in the PQ duration (P-R interval) (139%) than in the P-P interval (105%) at the last beat before the development of atrioventricular block. The prolongation in the P-P interval (11%, average 85 msec) and PQ duration during atrioventricular block disappeared immediately after the restoration of atrioventricular conduction. After edrophonium, the maximal prolongation in P-P (118%, p < 0.01) and PQ (120%, p < 0.01) intervals was the same. P-P interval remained prolonged (p < 0.01) after PQ interval returned to baseline. Neither ATP nor edrophonium affected the QRS duration. These findings suggest that ATP predominantly inhibited atrioventricular conduction rather than the firing rate of sinoatrial nodes, and edrophonium inhibited both proportionally even with prolonged inhibitory action on the sinoatrial nodes. An injection of ATP is needed only when a transient cardiac standstill is requested, such as in endovascular grafting surgery. Edrophonium may be used to slow heart rate during coronary artery bypass graft surgery. Copyright 2002, Elsevier Science (USA). All rights reserved.

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

    PubMed

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

    2016-03-15

    The novel, facile and universal aptamer-based methods for the highly sensitive and selective fluorescence detection of important biomolecules have attracted considerable interest. Here, we present a label-free aptasensor for adenosine triphosphate (ATP) detection in aqueous solutions by using an ultra-sensitive nucleic acid stain PicoGreen (PG) as a fluorescent indicator and core-shell Ag@SiO2 nanoparticles (NPs) as a metal-enhanced fluorescence (MEF) platform. In the presence of ATP, the complementary DNA (cDNA)/aptamer duplexes confined onto the Ag@SiO2 NPs surface can release their aptamers into the buffered solution, causing a significant reduction in fluorescence intensity. By virtue of the amplified fluorescence signal, this aptasensor toward ATP can achieve a detection limit of 14.2 nM with a wide linear range and exhibit a good assay performance in complex biological samples. This sensing approach is cost-effective and efficient because it avoids the fluorescence labeling process and the use of any enzymes. Hence, this method may offer an alternative tool for determining the concentrations of ATP in biochemical and biomedical research. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-10-01

    Adenosine 5'-triphosphate (ATP) is a potent signaling molecule that regulates diverse biological activities in cells. Its effects on human dental pulp cells (HDPCs) remain unknown. This study aimed to examine the effects of ATP on proliferation and differentiation of HDPCs. Reverse transcription polymerase chain reaction was performed to explore the mRNA expression of P2 receptor subtypes. Cell Counting Kit-8 test and flow cytometry analysis were used to examine the effects of ATP on proliferation and cell cycle of HDPCs. The effects of ATP on differentiation of HDPCs were examined by using alizarin red S staining, energy-dispersive x-ray analysis, Western blot analysis, and real-time polymerase chain reaction. The purinoceptors P2X3, P2X4, P2X5, P2X7, and all P2Y receptor subtypes were confirmed to present in HDPCs. ATP enhanced HDPC proliferation at 10 μmol/L concentration. However, it inhibited cell proliferation by arresting the cell cycle in G0G1 phase (P < .05 versus control) and induced odontoblastic differentiation, ERK/MAPK activation, and dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) mRNA transcriptions at 800 μmol/L concentration. Suramin, an ATP receptor antagonist, inhibited ERK/MAPK activation and HDPC odontoblastic differentiation (P < .05 versus control). Extracellular ATP activates P2 receptors and downstream signaling events that induce HDPC odontogenic differentiation. Thus, ATP may promote dental pulp tissue healing and repair through P2 signaling. Results provide new insights into the molecular regulation of pulpal wound healing. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2014-07-15

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

  15. Cyclic AMP differentiates two separate but interacting pathways of phosphoinositide hydrolysis in the DDT1-MF2 smooth muscle cell line.

    PubMed

    Schachter, J B; Wolfe, B B

    1992-03-01

    The activation of adenosine A1 receptors in DDT1-MF2 smooth muscle cells resulted in both the inhibition of agonist-stimulated cAMP accumulation and the potentiation of norepinephrine-stimulated phosphoinositide hydrolysis. Pharmacological analysis indicated the involvement of an A1 adenosine receptor subtype in both of these responses. In the absence of norepinephrine, the activation of the adenosine receptor did not directly stimulate phosphoinositide hydrolysis. The adenosine receptor-mediated augmentation of norepinephrine-stimulated phosphoinositide hydrolysis was pertussis toxin sensitive and was selectively antagonized by agents that mimicked cAMP (8-bromo-cAMP) or raised cellular cAMP levels (forskolin). This initially suggested that cAMP might partially regulate the magnitude of the phospholipase C response to norepinephrine and that adenosine agonists might enhance the phospholipase C response by reducing cAMP levels. However, neither the reduction of cellular cAMP levels by other agents nor the inhibition of cAMP-dependent protein kinase was sufficient to replicate the action of adenosine receptor activation on phosphoinositide hydrolysis. Thus, in the presence of norepinephrine, adenosine receptor agonists appear to stimulate phosphoinositide hydrolysis via a pathway that is separate from, but dependent upon, that of norepinephrine. This second pathway can be distinguished from that which is stimulated by norepinephrine on the basis of its sensitivity to inhibition by both cAMP and pertussis toxin.

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

    PubMed

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

    2016-01-01

    Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-05

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca(2+) signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca(2+) elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca(2+) signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5-10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca(2+) release followed by Ca(2+) entry and also substantially reduced Ca(2+) extrusion because of decreased intracellular ATP levels. The toxic Ca(2+) signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca(2+) signals and necrosis. We tested the effects of inhibiting the Ca(2+) release-activated Ca(2+) entry by the Ca(2+) channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca(2+) entry and also protected effectively against the development of necrosis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. © 2016 The Authors.

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

    PubMed

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

    2016-01-01

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

  1. Evaluation of the relationship between the Adenosine Triphosphate (ATP) bioluminescence assay and the presence of Bacillus anthracis spores and vegetative cells.

    PubMed

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

    2014-05-28

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

  2. Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

    PubMed

    Omori, Takashi; Idehara, Kenji; Kojima, Hajime; Sozu, Takashi; Arima, Kazunori; Goto, Hirohiko; Hanada, Tomohiko; Ikarashi, Yoshiaki; Inoda, Taketo; Kanazawa, Yukiko; Kosaka, Tadashi; Maki, Eiji; Morimoto, Takashi; Shinoda, Shinsuke; Shinoda, Naoki; Takeyoshi, Masahiro; Tanaka, Masashi; Uratani, Mamoru; Usami, Masahito; Yamanaka, Atsushi; Yoneda, Tomofumi; Yoshimura, Isao; Yuasa, Atsuko

    2008-01-01

    The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

  3. Role of hemolysis in red cell adenosine triphosphate release in simulated exercise conditions in vitro.

    PubMed

    Mairbäurl, Heimo; Ruppe, Florian A; Bärtsch, Peter

    2013-10-01

    Specific adenosine triphosphate (ATP) release from red blood cells has been discussed as a possible mediator controlling microcirculation in states of decreased tissue oxygen. Because intravascular hemolysis might also contribute to plasma ATP, we tested in vitro which portion of ATP release is due to hemolysis in typical exercise-induced strains to the red blood cells (shear stress, deoxygenation, and lactic acidosis). Human erythrocytes were suspended in dextran-containing media (hematocrit 10%) and were exposed to shear stress in a rotating Couette viscometer at 37°C. Desaturation (oxygen saturation of hemoglobin ∼20%) was achieved by tonometry with N2 before shear stress exposure. Cells not exposed to shear stress were used as controls. Na lactate (15 mM), lactic acid (15 mM, pH 7.0), and HCl (pH 7.0) were added to simulate exercise-induced lactic acidosis. After incubation, extracellular hemoglobin was measured to quantify hemolysis. ATP was measured with the luciferase assay. Shear stress increased extracellular ATP in a stress-related and time-dependent manner. Hypoxia induced a ∼10-fold increase in extracellular ATP in nonsheared cells and shear stress-exposed cells. Lactic acid had no significant effect on ATP release and hemolysis. In normoxic cells, approximately 20%-50% of extracellular ATP was due to hemolysis. This proportion decreased to less than 10% in hypoxic cells. Our results indicate that when exposing red blood cells to typical strains they encounter when passing through capillaries of exercising skeletal muscle, ATP release from red blood cells is caused mainly by deoxygenation and shear stress, whereas lactic acidosis had only a minor effect. Hemolysis effects were decreased when hemoglobin was deoxygenated. Together, by specific release and hemolysis, extracellular ATP reaches values that have been shown to cause local vasodilatation.

  4. Mucosal adenosine triphosphate mediates serotonin release from ileal but not colonic guinea pig enterochromaffin cells.

    PubMed

    Patel, B A

    2014-02-01

    Mechanical stimulation of the mucosal epithelium results in increased serotonin (5-HT) release from enterochromaffin (EC) cells. Little is known about how this process varies in different regions of the intestinal tract; however, purines are felt to play a role. We studied the relationship between mechanical stimulation, adenosine triphosphate (ATP), and 5-HT release from ileal and colonic mucosal tissue. Amperometric recordings of ATP and 5-HT were carried out using an ATP biosensor and boron-doped diamond microelectrode. Levels of extracellular ATP and 5-HT were monitored using high performance liquid chromatography. Under basal conditions, 5-HT levels were significantly decreased in the ileum (p < 0.001) but not the colon in the presence of the P2 antagonist suramin (100 μM). Ecto-ATPase inhibitor ARL67156 (10 μM) elevated ATP levels in the ileum and colon (both p < 0.001), but only 5-HT levels in the ileum (p < 0.001). Exogenous ATP increased 5-HT release in the presence of tetrodotoxin in the ileum (p < 0.001), but had not effect in the colon. Mechanical stimulation increased levels of 5-HT in the ileum (p < 0.001) and colon (p < 0.01), but levels returned to baseline in the presence of suramin and MRS2179 in the ileum. The onset of 5-HT release was delayed following mechanical stimulation. The rise time of the ATP response was quicker than that of 5-HT during mechanical stimulation. During mechanical stimulation of the mucosal epithelium, ATP mediates 5-HT release from EC cells in the ileum, but not the colon. Mucosal 5-HT signaling following mechanical stimulation is varied in different regions of the intestinal tract. © 2013 John Wiley & Sons Ltd.

  5. A High Affinity Adenosine Kinase from Anopheles gambiae

    PubMed Central

    Cassera, María B.; Ho, Meng-Chiao; Merino, Emilio F.; Burgos, Emmanuel S.; Rinaldo-Matthis, Agnes; Almo, Steven C.; Schramm, Vern L.

    2011-01-01

    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 (Km 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 Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises 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 human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

  6. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    M Cassera; M Ho; E Merino

    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 interactionsmore » 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.« less

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

    PubMed

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

    2011-08-17

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

  8. Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.

    PubMed

    Li, M; Marubayashi, A; Nakaya, Y; Fukui, K; Arase, S

    2001-12-01

    The mechanism by which minoxidil, an adenosine-triphosphate-sensitive potassium channel opener, induces hypertrichosis remains to be elucidated. Minoxidil has been reported to stimulate the production of vascular endothelial growth factor, a possible promoter of hair growth, in cultured dermal papilla cells. The mechanism of production of vascular endothelial growth factor remains unclear, however. We hypothesize that adenosine serves as a mediator of vascular endothelial growth factor production. Minoxidil-induced increases in levels of intracellular Ca(2+) and vascular endothelial growth factor production in cultured dermal papilla cells were found to be inhibited by 8-sulfophenyl theophylline, a specific antagonist for adenosine receptors, suggesting that dermal papilla cells possess adenosine receptors and sulfonylurea receptors, the latter of which is a well-known target receptor for adenosine-triphosphate-sensitive potassium channel openers. The expression of sulfonylurea receptor 2B and of the adenosine A1, A2A, and A2B receptors was detected in dermal papilla cells by means of reverse transcription polymerase chain reaction analysis. In order to determine which of the adenosine receptor subtypes contribute to minoxidil-induced hair growth, the effects of subtype-specific antagonists for adenosine receptors were investigated. Significant inhibition in increase in intracellular calcium level by minoxidil or adenosine was observed as the result of pretreatment with 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptor, but not by 3,7-dimethyl-1-propargyl-xanthine, an antagonist for adenosine A2 receptor, whereas vascular endothelial growth factor production was blocked by both adenosine A1 and A2 receptor antagonists. These results indicate that the effect of minoxidil is mediated by adenosine, which triggers intracellular signal transduction via both adenosine A1 and A2 receptors, and that the expression of sulfonylurea receptor 2B in

  9. A new s-adenosylhomocysteine hydrolase-linked method for adenosine detection based on DNA-templated fluorescent Cu/Ag nanoclusters.

    PubMed

    Ahn, Jun Ki; Kim, Hyo Yong; Baek, Songyi; Park, Hyun Gyu

    2017-07-15

    We herein describe a novel fluorescent method for the rapid and selective detection of adenosine by utilizing DNA-templated Cu/Ag nanoclusters (NCs) and employing s-adenosylhomocysteine hydrolase (SAHH). SAHH is allowed to promote hydrolysis reaction of s-adenosylhomocysteine (SAH) and consequently produces homocysteine, which would quench the fluorescence signal from DNA-templated Cu/Ag nanoclusters employed as a signaling probe in this study. On the other hand, adenosine significantly inhibits the hydrolysis reaction and prevent the formation of homocysteine. Consequently, highly enhanced fluorescence signal from DNA-Cu/Ag NCs is retained, which could be used to identify the presence of adenosine. By employing this design principle, adenosine was sensitively detected down to 19nM with high specificity over other adenosine analogs such as AMP, ADP, ATP, cAMP, guanosine, cytidine, and urine. Finally, the diagnostic capability of this method was successfully verified by reliably detecting adenosine present in a real human serum sample. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Poly(propyleneimine) glycodendrimers non-covalently bind ATP in a pH- and salt-dependent manner - model studies for adenosine analogue drug delivery.

    PubMed

    Gorzkiewicz, Michał; Buczkowski, Adam; Appelhans, Dietmar; Voit, Brigitte; Pułaski, Łukasz; Pałecz, Bartłomiej; Klajnert-Maculewicz, Barbara

    2018-06-10

    Adenosine analogue drugs (such as fludarabine or cladribine) require transporter-mediated uptake into cells and subsequent phosphorylation for anticancer activity. Therefore, application of nanocarrier systems for direct delivery of active triphosphate forms has been proposed. Here, we applied isothermal titration calorimetry and zeta potential titration to determine the stoichiometry and thermodynamic parameters of interactions between 4th generation poly(propyleneimine) dendrimers (unmodified or sugar-modified for increased biocompatibility) and ATP as a model adenosine nucleotide. We showed that glycodendrimers have the ability to efficiently interact with nucleoside triphosphates and to form stable complexes via electrostatic interactions between the ionized phosphate and amino groups on the nucleotide and the dendrimer, respectively. The complexation process is spontaneous, enthalpy-driven and depends on buffer composition (strongest interactions in organic buffer) and pH (more binding sites in acidic pH). These properties allow us to consider maltose-modified dendrimers as especially promising carriers for adenosine analogues. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. In Vitro Adenosine Triphosphate-Based Chemotherapy Response Assay as a Predictor of Clinical Response to Fluorouracil-Based Adjuvant Chemotherapy in Stage II Colorectal Cancer

    PubMed Central

    Kwon, Hye Youn; Kim, Im-kyung; Kang, Jeonghyun; Sohn, Seung-Kook; Lee, Kang Young

    2016-01-01

    Purpose We evaluated the usefulness of the in vitro adenosine triphosphate-based chemotherapy response assay (ATP-CRA) for prediction of clinical response to fluorouracil-based adjuvant chemotherapy in stage II colorectal cancer. Materials and Methods Tumor specimens of 86 patients with pathologically confirmed stage II colorectal adenocarcinoma were tested for chemosensitivity to fluorouracil. Chemosensitivity was determined by cell death rate (CDR) of drug-exposed cells, calculated by comparing the intracellular ATP level with that of untreated controls. Results Among the 86 enrolled patients who underwent radical surgery followed by fluorouracil-based adjuvant chemotherapy, recurrence was found in 11 patients (12.7%). The CDR ≥ 20% group was associated with better disease-free survival than the CDR < 20% group (89.4% vs. 70.1%, p=0.027). Multivariate analysis showed that CDR < 20% and T4 stage were poor prognostic factors for disease-free survival after fluorouracil-based adjuvant chemotherapy. Conclusion In stage II colorectal cancer, the in vitro ATP-CRA may be useful in identifying patients likely to benefit from fluorouracil-based adjuvant chemotherapy. PMID:26511802

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

    PubMed

    Pellegrini, Peter; Sauerwein, Rebecca; Finlayson, Tyler; McLeod, Jennifer; Covell, David A; Maier, Tom; Machida, Curtis A

    2009-04-01

    Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate (ATP)-driven bioluminescence could be used for rapid assessment of bacterial load in plaque. Patients (ages, 11-17 years) were bonded with SL and E brackets in 14 maxillary and 12 mandibular arches by using a split-mouth design. Recall visits were at 1 and 5 weeks after bonding. Plaque specimens were assayed for oral bacteria and subjected to ATP-driven bioluminescence determinations with a luciferin-based assay. In most patients, teeth bonded with SL attachments had fewer bacteria in plaque than did teeth bonded with E brackets. At 1 and 5 weeks after bonding, the means for SL vs E brackets were statistically lower for total bacteria and oral streptococci (P <0.05). ATP bioluminescence values were statistically correlated to the total oral bacteria and oral streptococci, with correlation coefficients of 0.895 and 0.843, respectively. SL appliances promote reduced retention of oral bacteria, and ATP bioluminescence might be a useful tool in the rapid quantification of bacterial load and the assessment of oral hygiene during orthodontic treatment.

  13. Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage.

    PubMed

    de Korte, Dirk; Kleine, Mya; Korsten, Herbert G H; Verhoeven, Arthur J

    2008-06-01

    Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (<0.2%). The 2,3-DPG content showed an increase until Day 21 (150% of initial level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.

  14. AMP is an adenosine A1 receptor agonist.

    PubMed

    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

    2012-02-17

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  15. The adenosine triphosphate test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels.

    PubMed

    Alfa, Michelle J; Fatima, Iram; Olson, Nancy

    2013-03-01

    The study objective was to verify that the adenosine triphosphate (ATP) benchmark of <200 relative light units (RLUs) was achievable in a busy endoscopy clinic that followed the manufacturer's manual cleaning instructions. All channels from patient-used colonoscopes (20) and duodenoscopes (20) in a tertiary care hospital endoscopy clinic were sampled after manual cleaning and tested for residual ATP. The ATP test benchmark for adequate manual cleaning was set at <200 RLUs. The benchmark for protein was <6.4 μg/cm(2), and, for bioburden, it was <4-log10 colony-forming units/cm(2). Our data demonstrated that 96% (115/120) of channels from 20 colonoscopes and 20 duodenoscopes evaluated met the ATP benchmark of <200 RLUs. The 5 channels that exceeded 200 RLUs were all elevator guide-wire channels. All 120 of the manually cleaned endoscopes tested had protein and bioburden levels that were compliant with accepted benchmarks for manual cleaning for suction-biopsy, air-water, and auxiliary water channels. Our data confirmed that, by following the endoscope manufacturer's manual cleaning recommendations, 96% of channels in gastrointestinal endoscopes would have <200 RLUs for the ATP test kit evaluated and would meet the accepted clean benchmarks for protein and bioburden. Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

  16. Correlation of Early Recurrence With In Vitro Adenosine Triphosphate Based Chemotherapy Response Assay in Pancreas Cancer With Postoperative Gemcitabine Chemotherapy.

    PubMed

    Park, Joon Seong; Kim, Jae Keun; Yoon, Dong Sup

    2016-11-01

    Gemcitabine-based regimens represent the standard systemic first line treatment in patients after pancreatic resection. However, the clinical impact of gemcitabine varies significantly in individuals because of chemoresistance. An in vitro adenosine triphosphate based chemotherapy response assay (ATP-CRA) was designed to evaluate the sensitivity of cancer cells to various chemotherapeutic agents. This study investigated the correlation between in vitro gemcitabine sensitivity of tumor cells and early recurrence after curative resection. From January 2007 to December 2010, the ATP-CRA for gemcitabine was tested in 64 patients surgically treated for pancreas cancer at Gangnam Severance Hospital, Seoul, Korea. We analyzed the relationship between chemosensitivity and early systemic recurrence in patients with pancreas cancer to predict disease-free survival (DFS) after curative resection in pancreas cancer. The mean cell death rate (CDR) was 20.0 (±14.5) and divided into two groups according to the mean values of the CDR. Lymphovascular invasion was more frequently shown in gemcitabine resistance group without statistical significance. In univariate and multivariate analysis, advanced tumor stage and gemcitabine sensitive group (CDR ≥ 20) were identified as independent prognostic factors for DFS. Gemcitabine sensitivity measured by ATP-CRA was well correlated with in vivo drug responsibility to predict early recurrence following gemcitabine-based adjuvant chemotherapy in patients with pancreas cancer. © 2016 Wiley Periodicals, Inc.

  17. Adenosine triphosphate bioluminescence for bacteriological surveillance and reprocessing strategies for minimizing risk of infection transmission by duodenoscopes

    PubMed Central

    Sethi, Saurabh; Huang, Robert J.; Barakat, Monique T.; Banaei, Niaz; Friedland, Shai; Banerjee, Subhas

    2017-01-01

    Background/Aims Recent outbreaks of duodenoscope-transmitted infections underscore the importance of adequate endoscope reprocessing. Adenosine triphosphate (ATP) bioluminescence testing allows rapid evaluation of endoscopes for bacteriological/biological residue. In this prospective study we evaluate the utility of ATP in bacteriological surveillance, and the effects of endoscopy staff education and dual cycles of cleaning and high-level disinfection (HLD) on endoscope reprocessing. Methods ATP bioluminescence was measured after pre-cleaning, manual cleaning and HLD on rinsates from suction-biopsy channels of all endoscopes and elevator channels of duodenoscopes/linear echoendoscopes after use. ATP bioluminescence was re-measured in duodenoscopes (1) after re-education and competency testing of endoscopy staff, and subsequently (2) after 2 cycles of pre-cleaning and manual cleaning and single cycle of HLD, or (3) after 2 cycles of pre-cleaning, manual cleaning and HLD. Results The ideal ATP bioluminescence benchmark of <200 relative light units (RLUs) after manual cleaning was achieved from suction-biopsy channel rinsates of all endoscopes, but 9 of 10 duodenoscope elevator channel rinsates failed to meet this benchmark. Re-education reduced RLUs in duodenoscope elevator channel rinsates after pre-cleaning (23218.0 vs 1340.5 RLUs, p<0.01) and HLD (177.0 vs 12.0 RLUs, p<0.01). After 2 cycles of manual cleaning/HLD, duodenoscope elevator channel RLUs achieved levels similar to sterile water, with corresponding negative cultures. Conclusions ATP testing offers a rapid, inexpensive alternative for detection of endoscope microbial residue. Re-education of endoscopy staff and 2 cycles of cleaning and HLD decrease elevator channel RLUs to levels similar to sterile water and may therefore minimize the risk of transmission of infections by duodenoscopes. PMID:27818222

  18. Determination of adenosine phosphates in rat gastrocnemius at various postmortem intervals using high performance liquid chromatography.

    PubMed

    Huang, Hong; Yan, Youyi; Zuo, Zhong; Yang, Lin; Li, Bin; Song, Yu; Liao, Linchuan

    2010-09-01

    Although the change in adenosine phosphate levels in muscles may contribute to the development of rigor mortis, the relationship between their levels and the onset and development of rigor mortis has not been well elucidated. In the current study, levels of the adenosine phosphates including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in gastrocnemius at various postmortem intervals of 180 rats from different death modes were detected by high performance liquid chromatography. The results showed that the levels of ATP and ADP significantly decreased along with the postmortem period of rats from different death mode whereas the AMP level remained the same. In addition, it was found that changes in the ATP levels in muscles after death correlated well with the development of rigor mortis. Therefore, the ATP level could serve as a reference parameter for the deduction of rigor mortis in forensic science.

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2013-06-01

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

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

    PubMed

    Flitney, F W; Singh, J

    1980-07-01

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

  2. AMP Is an Adenosine A1 Receptor Agonist*

    PubMed Central

    Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2012-01-01

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

  3. Repression of adenosine triphosphate-binding cassette transporter ABCG2 by estrogen increases intracellular glutathione in brain endothelial cells following ischemic reperfusion injury.

    PubMed

    Shin, Jin A; Jeong, Sae Im; Kim, Hye Won; Jang, Gyeonghui; Ryu, Dong-Ryeol; Ahn, Young-Ho; Choi, Ji Ha; Choi, Youn-Hee; Park, Eun-Mi

    2018-06-01

    The adenosine triphosphate-binding cassette efflux transporter ABCG2, which is located in the blood-brain barrier limits the entry of endogenous compounds and xenobiotics into the brain, and its expression and activity are regulated by estrogen. This study was aimed to define the role of ABCG2 in estrogen-mediated neuroprotection against ischemic injury. ABCG2 protein levels before and after ischemic stroke were increased in the brain of female mice by ovariectomy, which were reversed by estrogen replacement. In brain endothelial cell line bEnd.3, estrogen reduced the basal ABCG2 protein level and efflux activity and protected cells from ischemic injury without inducing ABCG2 expression. When bEnd.3 cells were transfected with ABCG2 small interfering RNA, ischemia-induced cell death was reduced, and the intracellular concentration of glutathione, an antioxidant that is transported by ABCG2, was increased. In addition, after ischemic stroke in ovariectomized mice, estrogen prevented the reduction of intracellular glutathione level in brain microvessels. These data suggested that the suppression of ABCG2 by estrogen is involved in neuroprotection against ischemic injury by increasing intracellular glutathione, and that the modulation of ABCG2 activity offers a therapeutic target for brain diseases in estrogen-deficient aged women. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2017-10-01

    Sepsis is a potentially lethal condition, and it is associated with platelet alterations. The present study sought to investigate the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA) in the platelets of rats that were induced with sepsis. Male Wistar rats were divided into three groups of ten animals each: a negative control group (normal; NC); a group that underwent surgical procedures (sham); and a group that underwent cecal ligation and perforation (CLP). The induction of sepsis was confirmed by bacteremia, and the causative pathogen identified was Escherichia coli. Hematological parameters showed leukocytosis and thrombocytopenia in animals in the septic group. The results also revealed that there were significant (p < 0.05) increases in adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolyses, and in the deamination of adenosine in the CLP group compared to the sham and control groups. Conversely, ADP hydrolysis was significantly decreased (p < 0.05) in the CLP group compared to the sham and control groups. Purine levels were analyzed by high-performance liquid chromatography (HPLC) in serum samples from control, sham, and CLP groups. Increased concentrations of ATP, adenosine, and inosine were found in the CLP group compared to the sham and control groups. Conversely, the concentrations of ADP and AMP in the CPL group were not significantly altered. We suggest that alterations in hematological parameters, nucleotide hydrolysis in platelets, and nucleotide concentrations in serum samples of rats with induced sepsis may be related to thromboembolic events. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Neurochemical Measurement of Adenosine in Discrete Brain Regions of Five Strains of Inbred Mice

    PubMed Central

    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

  6. [A prospective study of adenosine triphosphate-tumor chemosensitivity assay directed chemotherapy in patients with recurrent ovarian cancer].

    PubMed

    Gao, Yu-tao; Wu, Ling-ying; Zhang, Wei; Zhao, Dan; Li, Ning; Tian, Hai-mei; Wang, Xiao-bing; Li, Mo; Sun, Yang-chun; Li, Nan; Li, Xiao-guang

    2013-05-01

    To investigate the efficacy of adenosine triphosphate (ATP)-tumor chemosensitivity assay (TCA) directed chemotherapy in patients with recurrent epithelial ovarian cancer. From August 2010 to June 2012, recurrent epithelial ovarian cancer patients were prospectively enrollmented in Cancer Hospital, Peking Union Medical College,Chinese Academy of Medical Sciences.The entry criteria are as follows: (1) Histologically proven to be epithelial ovarian cancer. (2) Patients of recurrent ovarian cancer with bidimensionally measurable tumor, or ascitic or pleural fluid for testing. (3) Karnofsky performance status > 60. (4) A life expectancy of at least more than 6 months.According to patients desires, they were assigned into two groups: assay-directed therapy group and physician's-choice therapy group, patients' clinical and pathological characteristics, response rate to chemotherapy and progression-free survival (PFS) were compared between two groups. A total of 113 patients with recurrent epithelial ovarian cancer were prospectively enrollmented to assay-directed chemotherapy (n = 56) or physician's-choice chemotherapy (n = 57).There was no difference in median age,types of recurrence, surgical-pathological stage, pathological type, tumor grade, times of recurrence, residual disease at secondary cytoreductive surgery between assay-directed group and physician's-choice group. The overall response rate (ORR) and median PFS in the ATP-TCA group was 66% (37/56) and 7 months, while the ORR in the control group was 46% (26/57, P = 0.037), the median PFS was 4 months (P = 0.040). For platinum-resistant patients, the ORR between ATP-TCA directed chemotherapy 59% (16/27) and control group 25% (7/28) were significantly different (P = 0.010), and the median PFS between two groups were also significantly different (5 months and 2 months, respectively, P = 0.003). ATP-TCA directed chemotherapy could improve ORR and PFS in patients with recurrent epithelial ovarian cancer, especially

  7. The adenosine triphosphate method as a quality control tool to assess 'cleanliness' of frequently touched hospital surfaces.

    PubMed

    Knape, L; Hambraeus, A; Lytsy, B

    2015-10-01

    The adenosine triphosphate (ATP) method is widely accepted as a quality control method to complement visual assessment, in the specifications of requirements, when purchasing cleaning contractors in Swedish hospitals. To examine whether the amount of biological load, as measured by ATP on frequently touched near-patient surfaces, had been reduced after an intervention; to evaluate the correlation between visual assessment and ATP levels on the same surfaces; to identify aspects of the performance of the ATP method as a tool in evaluating hospital cleanliness. A prospective intervention study in three phases was carried out in a medical ward and an intensive care unit (ICU) at a regional hospital in mid-Sweden between 2012 and 2013. Existing cleaning procedures were defined and baseline tests were sampled by visual inspection and ATP measurements of ten frequently touched surfaces in patients' rooms before and after intervention. The intervention consisted of educating nursing staff about the importance of hospital cleaning and direct feedback of ATP levels before and after cleaning. The mixed model showed a significant decrease in ATP levels after the intervention (P < 0.001). Relative light unit values were lower in the ICU. Cleanliness as judged by visual assessments improved. In the logistic regression analysis, there was a significant association between visual assessments and ATP levels. Direct feedback of ATP levels, together with education and introduction of written cleaning protocols, were effective tools to improve cleanliness. Visual assessment correlated with the level of ATP but the correlation was not absolute. The ATP method could serve as an educational tool for staff, but is not enough to assess hospital cleanliness in general as only a limited part of a large area is covered. Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  8. Adenosine triphosphate bioluminescence for bacteriologic surveillance and reprocessing strategies for minimizing risk of infection transmission by duodenoscopes.

    PubMed

    Sethi, Saurabh; Huang, Robert J; Barakat, Monique T; Banaei, Niaz; Friedland, Shai; Banerjee, Subhas

    2017-06-01

    Recent outbreaks of duodenoscope-transmitted infections underscore the importance of adequate endoscope reprocessing. Adenosine triphosphate (ATP) bioluminescence testing allows rapid evaluation of endoscopes for bacteriologic/biologic residue. In this prospective study we evaluate the utility of ATP in bacteriologic surveillance and the effects of endoscopy staff education and dual cycles of cleaning and high-level disinfection (HLD) on endoscope reprocessing. ATP bioluminescence was measured after precleaning, manual cleaning, and HLD on rinsates from suction-biopsy channels of all endoscopes and elevator channels of duodenoscopes/linear echoendoscopes after use. ATP bioluminescence was remeasured in duodenoscopes (1) after re-education and competency testing of endoscopy staff and subsequently (2) after 2 cycles of precleaning and manual cleaning and single cycle of HLD or (3) after 2 cycles of precleaning, manual cleaning, and HLD. The ideal ATP bioluminescence benchmark of <200 relative light units (RLUs) after manual cleaning was achieved from suction-biopsy channel rinsates of all endoscopes, but 9 of 10 duodenoscope elevator channel rinsates failed to meet this benchmark. Re-education reduced RLUs in duodenoscope elevator channel rinsates after precleaning (23,218.0 vs 1340.5 RLUs, P < .01) and HLD (177.0 vs 12.0 RLUs, P < .01). After 2 cycles of manual cleaning/HLD, duodenoscope elevator channel RLUs achieved levels similar to sterile water, with corresponding negative cultures. ATP testing offers a rapid, inexpensive alternative for detection of endoscope microbial residue. Re-education of endoscopy staff and 2 cycles of cleaning and HLD decreased elevator channel RLUs to levels similar to sterile water and may therefore minimize the risk of transmission of infections by duodenoscopes. Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

  9. Adenosine triphosphate diffusion through poly(ethylene glycol) diacrylate hydrogels can be tuned by cross-link density as measured by PFG-NMR

    NASA Astrophysics Data System (ADS)

    Majer, Günter; Southan, Alexander

    2017-06-01

    The diffusion of small molecules through hydrogels is of great importance for many applications. Especially in biological contexts, the diffusion of nutrients through hydrogel networks defines whether cells can survive inside the hydrogel or not. In this contribution, hydrogels based on poly(ethylene glycol) diacrylate with mesh sizes ranging from ξ = 1.1 to 12.9 nm are prepared using polymers with number-average molecular weights between Mn = 700 and 8000 g/mol. Precise measurements of diffusion coefficients D of adenosine triphosphate (ATP), an important energy carrier in biological systems, in these hydrogels are performed by pulsed field gradient nuclear magnetic resonance. Depending on the mesh size, ξ, and on the polymer volume fraction of the hydrogel after swelling, ϕ, it is possible to tune the relative ATP diffusion coefficient D/D0 in the hydrogels to values between 0.14 and 0.77 compared to the ATP diffusion coefficient D0 in water. The diffusion coefficients of ATP in these hydrogels are compared with predictions of various mathematical expressions developed under different model assumptions. The experimental data are found to be in good agreement with the predictions of a modified obstruction model or the free volume theory in combination with the sieving behavior of the polymer chains. No reasonable agreement was found with the pure hydrodynamic model.

  10. Increased adenosine triphosphate production by peripheral blood CD4+ cells in patients with hematologic malignancies treated with stem cell mobilization agents.

    PubMed

    Manga, Kiran; Serban, Geo; Schwartz, Joseph; Slotky, Ronit; Patel, Nita; Fan, Jianshe; Bai, Xiaolin; Chari, Ajai; Savage, David; Suciu-Foca, Nicole; Colovai, Adriana I

    2010-07-01

    Hematopoietic stem cell (HSC) transplantation is an important therapeutic option for patients with hematologic malignancies. To explore the immunomodulatory effects of HSC mobilization agents, we studied the function and phenotype of CD4(+) T cells from 16 adult patients with hematologic malignancies undergoing HSC mobilization treatment for autologous transplantation. Immune cell function was determined using the Immuknow (Cylex) assay by measuring the amount of adenosine triphosphate (ATP) produced by CD4(+) cells from whole blood. ATP activity measured in G-CSF-treated patients was significantly higher than that measured in healthy individuals or "nonmobilized" patients. In patients treated with G-CSF, CD4(+) T cells were predominantly CD25(low)FOXP3(low), consistent with an activated phenotype. However, T-cell depletion did not abrogate ATP production in blood samples from G-CSF-treated patients, indicating that CD4(+) myeloid cells contributed to the increased ATP levels observed in these patients. There was a significant correlation between ATP activity and patient survival, suggesting that efficient activation of CD4(+) cells during mobilization treatment predicts a low risk of disease relapse. Monitoring immune cell reactivity using the Immuknow assay may assist in the clinical management of patients with hematologic malignancies and optimization of HSC mobilization protocols. Copyright 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  11. Yolk-Shell Porous Microspheres of Calcium Phosphate Prepared by Using Calcium L-Lactate and Adenosine 5'-Triphosphate Disodium Salt: Application in Protein/Drug Delivery.

    PubMed

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

    2015-06-26

    A facile and environmentally friendly approach has been developed to prepare yolk-shell porous microspheres of calcium phosphate by using calcium L-lactate pentahydrate (CL) as the calcium source and adenosine 5'-triphosphate disodium salt (ATP) as the phosphate source through the microwave-assisted hydrothermal method. The effects of the concentration of CL, the microwave hydrothermal temperature, and the time on the morphology and crystal phase of the product are investigated. The possible formation mechanism of yolk-shell porous microspheres of calcium phosphate is proposed. Hemoglobin from bovine red cells (Hb) and ibuprofen (IBU) are used to explore the application potential of yolk-shell porous microspheres of calcium phosphate in protein/drug loading and delivery. The experimental results indicate that the as-prepared yolk-shell porous microspheres of calcium phosphate have relatively high protein/drug loading capacity, sustained protein/drug release, favorable pH-responsive release behavior, and a high biocompatibility in the cytotoxicity test. Therefore, the yolk-shell porous microspheres of calcium phosphate have promising applications in various biomedical fields such as protein/drug delivery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Evaluation of adenosine triphosphate test for cleaning assessment of gastroscopes and the effect on workload in a busy endoscopy center.

    PubMed

    Schmitt, Cristiane; Pires Maciel, Amanda Luiz; Boszczowski, Icaro; da Silva, Thaís Pereira; Neves, Eliane Aparecida Job; Rossini, Giulio Fabio; Rizek, Camila; Costa, Silvia Figueiredo; Lourenço, Rogério Ferreira; Alfa, Michelle J

    2018-05-18

    Using adenosine triphosphate (ATP) tests to assess manual cleaning of gastroscopes and to determine the associated workload in a busy endoscopy unit. Patient-used gastroscopes were sampled before and after cleaning to assess ATP levels, bioburden, and protein. Samples were collected by flushing 20 mL of sterile water through the biopsy port to the distal end. Time spent for reprocessing and performing the ATP test was recorded. Twenty-four samples were collected from 10 gastroscopes. After manual cleaning, 14/24 (58.3%) samples had no microbial growth (mean, 21 colony-forming units/cm 2 ), and in 22/24 (91.7%) samples the protein was undetectable (mean, 0.04 µg/cm 2 ). ATP test was above the cutoff (200 relative light units [RLU]) in 17/24 (70.8%) samples (mean, 498 RLU). After the second cleaning, 11/17 (64.7%) gastroscopes still failed the ATP test (mean, 321.2 RLU). The mean time spent to perform manual cleaning and ATP tests was 16 and 8 minutes, respectively. Hence, each test increased the length of time for cleaning plus testing cleanliness by 50%. Further studies regarding the optimal cutoff for ATP tests are needed. ATP tests for cleaning monitoring are easy to perform and provide immediate feedback to the team. However, the increased workload needs to be considered. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

  13. A novel inositol phosphate selectively inhibits vasoconstriction evoked by the sympathetic co-transmitters neuropeptide Y (NPY) and adenosine triphosphate (ATP).

    PubMed

    Wahlestedt, C; Reis, D J; Yoo, H; Adamsson, M; Andersson, D; Edvinsson, L

    1992-08-31

    Postganglionic sympathetic nerves release norepinephrine (NE) as their primary neurotransmitter at vascular and other targets. However, much evidence supports involvement of additional messengers, co-transmitters, which are co-released with NE upon sympathetic nerve stimulation and thereby contribute to their actions, e.g., vasoconstriction. Two such putative co-transmitters, neuropeptide Y (NPY) and adenosine triphosphate (ATP) have been of particular interest since they fulfill several neurotransmitter criteria. Importantly, hitherto it has been difficult to antagonize vasoconstriction evoked by either NPY or ATP with agents that are devoid of intrinsic activity. The present study describes the ability of a novel inositol phosphate, D-myo-inositol 1,2,6-trisphosphate (Ins[1,2,6]P3; PP-56) to in vitro potently block vasoconstrictor responses elicited by NPY and ATP, but not by NE, as studied in guinea-pig isolated basilar artery. The action of Ins[1,2,6]P3 does not seem to occur through antagonism at NPY- or ATP-receptor recognition sites, labeled by 125I-peptide YY and 35S-gamma-ATP, respectively, in membranes of rat cultured vena cava vascular smooth muscle cells. However, it does involve inhibition of the influx of Ca2+ induced by either co-transmitter in these same vena cava cells. It is proposed that Ins[1,2,6]P3 may be a useful functional antagonist of non-adrenergic component(s) of the vasoconstrictor response to sympathetic nerve stimulation.

  14. An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

    PubMed

    Rubinstein, D; Warrendorf, E

    1975-06-01

    The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.

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

    PubMed

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

    2013-10-01

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

  16. Differential distribution of adenosine receptors in rat cochlea.

    PubMed

    Vlajkovic, Srdjan M; Abi, Shukri; Wang, Carol J H; Housley, Gary D; Thorne, Peter R

    2007-06-01

    Adenosine is a constitutive cell metabolite that can be released from cells via specific bi-directional transporters and is an end-point for nucleotide hydrolysis. In the extracellular space, adenosine becomes a signalling molecule for P1 (adenosine) receptors that modulate physiological responses in a wide range of mammalian tissues. Whereas adenosine signalling has been implicated in the regulation of cochlear blood flow and in cochlear protection from oxidative damage, the potential roles for adenosine signalling in the modulation of sound transduction and auditory neurotransmission have not been established. We have characterised the expression and distribution of adenosine receptors in the rat cochlea. mRNA transcripts for all four subtypes of adenosine receptors (A(1), A(2A), A(2B) and A(3)) were detected in dissected cochlear tissue by using reverse transcription/polymerase chain reaction analysis. The protein distribution for the A(1), A(2A) and A(3) receptor subtypes was identified by immunoperoxidase histochemistry and confocal immunofluorescence labelling. These receptors were differentially expressed in the organ of Corti, spiral ganglion neurones, lateral wall tissues and cochlear blood vessels. The distribution of adenosine receptors in sensory and neural tissues and in the vasculature coincided with other elements of purinergic signalling (P2X and P2Y receptors, ectonucleotidases), consistent with the integrative regulation of many physiological processes in the cochlea by extracellular nucleotides and nucleosides. Our study provides a framework for further investigation of adenosine signalling in the inner ear, including putative roles in oxidative stress responses.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    1991-01-01

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

  18. A fluorescent aptasensor for analysis of adenosine triphosphate based on aptamer-magnetic nanoparticles and its single-stranded complementary DNA labeled carbon dots.

    PubMed

    Saberi, Zeinab; Rezaei, Behzad; Khayamian, Taghi

    2018-06-01

    A new fluorimetric aptasensor was designed for the determination of adenosine triphosphate (ATP) based on magnetic nanoparticles (MNPs) and carbon dots (CDs). In this analytical strategy, an ATP aptamer was conjugated on MNPs and a complementary strand of the aptamer (CS) was labeled with CDs. The aptamer and its CS were hybridized to form a double helical structure. The hybridized aptamers could be used for the specific recognition of ATP in a biological complex matrix using a strong magnetic field to remove the interfering effect. In the absence of ATP, no CDs-CS could be released into the solution and this resulted in a weak fluorescence signal. In the presence of ATP, the target binds to its aptamer and causes the dissociation of the double helical structure and liberation of the CS, such that a strong fluorescence signal was generated. The increased fluorescence signal was proportional to ATP concentration. The limit of detection was estimated to be 1.0 pmol L -1 with a dynamic range of 3.0 pmol L -1 to 5.0 nmol L -1 . The specific aptasensor was applied to detect ATP in human serum samples with satisfactory results. Moreover, molecular dynamic simulation (MDS) studies were used to analyze interactions of the ATP molecule with the aptamer. Copyright © 2018 John Wiley & Sons, Ltd.

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

    PubMed

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

    2016-06-09

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

  20. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

    PubMed

    Aschenbach, William G; Sakamoto, Kei; Goodyear, Laurie J

    2004-01-01

    The 5' adenosine monophosphate-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that functions as a metabolic 'fuel gauge' in skeletal muscle. AMPK is a ubiquitous heterotrimeric protein, consisting of an alpha catalytic, and beta and gamma regulatory subunits that exist in multiple isoforms and are all required for full enzymatic activity. During exercise, AMPK becomes activated in skeletal muscle in response to changes in cellular energy status (e.g. increased adenosine monophosphate [AMP]/adenosine triphosphate [ATP] and creatine/phosphocreatine ratios) in an intensity-dependent manner, and serves to inhibit ATP-consuming pathways, and activate pathways involved in carbohydrate and fatty-acid metabolism to restore ATP levels. Recent evidence shows that although AMPK plays this key metabolic role during acute bouts of exercise, it is also an important component of the adaptive response of skeletal muscles to endurance exercise training because of its ability to alter muscle fuel reserves and expression of several exercise-responsive genes. This review discusses the putative roles of AMPK in acute and chronic exercise responses, and suggests avenues for future AMPK research in exercise physiology and biochemistry.

  1. Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

    PubMed

    Proszkowiec-Weglarz, M; Richards, M P

    2009-01-01

    The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

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

    PubMed

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

    2014-08-07

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

  3. Hepatic fat and adenosine triphosphate measurement in overweight and obese adults using 1H and 31P magnetic resonance spectroscopy

    PubMed Central

    Solga, Steven F.; Horska, Alena; Hemker, Susanne; Crawford, Stephen; Diggs, Charalett; Diehl, Anna Mae; Brancati, Frederick L.; Clark, Jeanne M.

    2009-01-01

    Background/Aims Magnetic resonance spectroscopy (MRS) measures hepatic fat and adenosine triphosphate (ATP), but magnetic resonance studies are challenging in obese subjects. We aimed to evaluate the inter- and intrarater reliability and stability of hepatic fat and ATP measurements in a cohort of overweight and obese adults. Methods We measured hepatic fat and ATP using proton MRS (1H MRS) and phosphorus MRS (31P MRS) at baseline in adults enrolled in the Action for Health in Diabetes (Look AHEAD) clinical trial at one site. Using logistic regression, we determined factors associated with successful MRS data acquisition. We calculated the intra- and inter-rater reliability for hepatic fat and ATP based on 20 scans analysed twice by two readers. We also calculated the stability of these measures three times on five healthy volunteers. Results Of 244 participants recruited into our ancillary study, 185 agreed to MRS. We obtained usable hepatic fat data from 151 (82%) and ATP data from 105 (58%). Obesity was the strongest predictor of failed data acquisition; every unit increase in the body mass index reduced the likelihood of successful fat data by 11% and ATP data by 14%. The inter- and intrarater reliability were excellent for fat (intraclass correlation coefficient = 0.99), but substantially more variable for ATP. Fat measures appeared relatively stable, but this was less true for ATP. Conclusions Obesity can hinder 1H and 31P MRS data acquisition and subsequent analysis. This impact was greater for hepatic ATP than hepatic fat. PMID:18331237

  4. 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

  5. Endogenous Production of Extracellular Adenosine by Trabecular Meshwork Cells: Potential Role in Outflow Regulation

    PubMed Central

    Wu, Jing; Li, Guorong; Luna, Coralia; Spasojevic, Ivan; Epstein, David L.; Gonzalez, Pedro

    2012-01-01

    Purpose. To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. Methods. Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-β-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. Results. PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. Conclusions. These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma. PMID:22997289

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

    PubMed

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

    2014-02-01

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

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

    PubMed

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

    2016-06-13

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

  8. Amplification of the effects of magnetization exchange by (31) P band inversion for measuring adenosine triphosphate synthesis rates in human skeletal muscle.

    PubMed

    Ren, Jimin; Sherry, A Dean; Malloy, Craig R

    2015-12-01

    The goal of this study was to amplify the effects of magnetization exchange between γ-adenosine triphosphate (ATP) and inorganic phosphate (Pi) for evaluation of ATP synthesis rates in human skeletal muscle. The strategy works by simultaneously inverting the (31) P resonances of phosphocreatine (PCr) and ATP using a wide bandwidth, adiabatic inversion radiofrequency pulse followed by observing dynamic changes in intensity of the noninverted Pi signal versus the delay time between the inversion and observation pulses. This band inversion technique significantly delays recovery of γ-ATP magnetization; consequently, the exchange reaction, Pi ↔ γ-ATP, is readily detected and easily analyzed. The ATP synthesis rate measured from high-quality spectral data using this method was 0.073 ± 0.011 s(-1) in resting human skeletal muscle (N = 10). The T1 of Pi was 6.93 ± 1.90 s, consistent with the intrinsic T1 of Pi at this field. The apparent T1 of γ-ATP was 4.07 ± 0.32 s, about two-fold longer than its intrinsic T1 due to storage of magnetization in PCr. Band inversion provides an effective method to amplify the effects of magnetization transfer between γ-ATP and Pi. The resulting data can be easily analyzed to obtain the ATP synthesis rate using a two-site exchange model. © 2014 Wiley Periodicals, Inc.

  9. Reconsideration of the sequence of rigor mortis through postmortem changes in adenosine nucleotides and lactic acid in different rat muscles.

    PubMed

    Kobayashi, M; Takatori, T; Iwadate, K; Nakajima, M

    1996-10-25

    We examined the changes in adenosine triphosphate (ATP), lactic acid, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in five different rat muscles after death. Rigor mortis has been thought to occur simultaneously in dead muscles and hence to start in small muscles sooner than in large muscles. In this study we found that the rate of decrease in ATP was significantly different in each muscle. The greatest drop in ATP was observed in the masseter muscle. These findings contradict the conventional theory of rigor mortis. Similarly, the rates of change in ADP and lactic acid, which are thought to be related to the consumption or production of ATP, were different in each muscle. However, the rate of change of AMP was the same in each muscle.

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

    PubMed

    Akintunde, J K; Oboh, G

    2015-11-01

    One of the major hazards arising from recycling sites is the generation of leachate containing mixed metal. This study evaluated the toxic effects of leachate obtained from Elewi Odo municipal auto-battery recycling site (EOMABRSL) on male liver functions using hepatic indices and biomarker of cellular adenosine triphosphate (ATP) in rat via the oral route. Concentrations of heavy metals analysis showed that lead, cadmium, nickel, chromium, manganese, and iron were 1.5-, 2-, 2.5-, 1.36-, 19.61-, and 8.89-folds, respectively, higher than acceptable limits set by regulatory authority World Health Organization. Copper, zinc, and cobalt were 5.9-, 300-, and 1.02-folds, respectively, lower than permissible limits. The EOMABRSL was administered at 20, 40, 60, 80, and 100% concentrations to adult male rats for 60 days. Following exposure, plasma and livers were collected for several biochemistry assays. Exposure of animals to EOMABRSL resulted in 27.51, 28.14, 63.93, 28.42, and 40.16% increase in aspartate aminotransferase activity, whereas it elevated alanine aminotransferase activity by 5.35, 22.33, 88.68, 183.02, and 193.08%, respectively, when compared with the control. Similarly, γ-glutamyl transferase activity increased by 111.22, 114.19, 122.96, 573.14, and 437.02%, respectively, when compared with the control. EOMABRSL administration significantly decreased catalase activity and reduced glutathione level and superoxide dismutase with concomitant increase in malondialdehyde and hydrogen peroxide levels. Also, significant (p < 0.05) decrease in lactate dehydrogenase (LDH) activity (marker of cellular ATP) was observed. Taken together, the hepatotoxicity of EOMABRSL could be due to the depletion of LDH and induction of oxidative damage, which may suggest possible health hazards in subjects with occupational or environmental exposure. © The Author(s) 2015.

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

    PubMed Central

    Sazani, Peter L.; Larralde, Rosa

    2004-01-01

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

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

    PubMed

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

    2017-01-29

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors.

    PubMed Central

    Huang, N; Wang, D J; Heppel, L A

    1989-01-01

    Extracellular ATP in concentrations of 5-50 microM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl)carboxamidoadenosine. Cultures of Swiss 3T3, Swiss 3T6, A431, DDT1-MF2, and HFF cells were used. The percent of cell nuclei labeled with [3H]thymidine and cell number were also increased. ADP was equally mitogenic, while UTP and ITP were much less active. The effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine, a known growth factor. Thus, the nonhydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate was mitogenic. In addition, it was found that ATP showed synergism in 3T6 and 3T3 cells when present for only the first hour of an incorporation assay, during which time no significant hydrolysis occurred. Furthermore, prolonged preincubation of cells with ATP reduced the mitogenic response to ATP but not to adenosine; preincubation with adenosine or N6-(R-phenylisopropyl)adenosine had the reverse effect. Finally, the effect of adenosine, but not of ATP, was inhibited by aminophylline. We conclude that extracellular ATP is a mitogen that interacts with P2 purinoceptors on the plasma membrane. PMID:2813367

  16. Central or Peripheral Delivery of an Adenosine A1 Receptor Agonist Improves Mechanical Allodynia in a Mouse Model of Painful Diabetic Neuropathy

    PubMed Central

    Katz, N. K.; Ryals, J. M.; Wright, D. E.

    2014-01-01

    Diabetic peripheral neuropathy is a common complication of diabetes mellitus, and a significant proportion of individuals suffer debilitating pain that significantly affects their quality of life. Unfortunately, symptomatic treatment options have limited efficacy, and often carry significant risk of systemic adverse effects. Activation of the adenosine A1 receptor (A1R) by the analgesic small molecule adenosine has been shown to have antinociceptive benefits in models of inflammatory and neuropathic pain. The current study used a mouse model of painful diabetic neuropathy to determine the effect of diabetes on endogenous adenosine production, and if central or peripheral delivery of adenosine receptor agonists could alleviate signs of mechanical allodynia in diabetic mice. Diabetes was induced using streptozocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Hydrolysis of AMP into adenosine by ectonucleotidases was determined in the dorsal root ganglia (DRG) and spinal cord at 8-weeks post-induction of diabetes. AMP, adenosine and the specific A1R agonist, N6-cyclopentyladenosine (CPA), were administered both centrally (intrathecal) and peripherally (intraplantar) to determine the effect of activation of adenosine receptors on mechanical allodynia in diabetic mice. Eight weeks post-induction, diabetic mice displayed significantly decreased hydrolysis of extracellular AMP in the DRG; at this same time, diabetic mice displayed significantly decreased mechanical withdrawal thresholds compared to nondiabetic controls. Central delivery AMP, adenosine and CPA significantly improved mechanical withdrawal thresholds in diabetic mice. Surprisingly, peripheral delivery of CPA also improved mechanical allodynia in diabetic mice. This study provides new evidence that diabetes significantly affects endogenous AMP hydrolysis, suggesting that altered adenosine production could contribute to the development of painful

  17. Functional effects of uridine triphosphate on human skinned skeletal muscle fibers.

    PubMed

    Vianna-Jorge, R; Oliveira, C F; Mounier, Y; Suarez-Kurtz, G

    1998-02-01

    Chemically skinned human skeletal muscle fibers were used to study the effects of uridine triphosphate (UTP) on the tension-pCa relationship and on Ca2+ uptake and release by the sarcoplasmic reticulum (SR). Total replacement (2.5 mM) of adenosine triphosphate (ATP) with UTP (i) displaced the tension-pCa relationship to the left along the abcissae and increased maximum Ca(2+)-activated tension, both effects being larger in slow- than in fast-type fibers; (ii) markedly reduced Ca2+ uptake by the SR (evaluated by the caffeine-evoked tension) in both fiber types; (iii) had no effect on the rate of depletion of caffeine-sensitive Ca2+ stores during soaking in relaxing solutions; (iv) induced tension in slow- but not in fast-type fibers. The effects on the SR functional properties are consistent with the notion that UTP is a poor substitute for ATP as a substrate for the Ca ATPase pump and as an agonist of the ryanodine-sensitive Ca(2+)-release channel. The UTP-induced tension in human slow-type fibers is attributed to effect(s) of the nucleotide on the tension-pCa relationship of the contractile machinery. The present data reveal important differences between the effects of UTP on human versus rat muscle fibers.

  18. Aprikalim a potassium adenosine triphosphate channel opener reduces neurologic injury in a rabbit model of spinal cord ischemia.

    PubMed

    Lozos, Vasileios A; Toumpoulis, Ioannis K; Agrogiannis, Georgios; Giamarellos-Bourboulis, Evangelos J; Chamogeorgakis, Themistocles P; Rizos, Ioannis K; Patsouris, Efstratios S; Anagnostopoulos, Constantine E; Rokkas, Chris K

    2013-01-01

    Potassium adenosine triphosphate (KATP) channel openers have been involved in the enhancement of ischemic tolerance in various tissues. The purpose of the present study is to evaluate the effects of aprikalim, a specific KATP channel opener, on spinal cord ischemic injury. Fifty-four rabbits were randomly assigned to three groups: group 1 (n = 18, sham operation), group 2 (n = 18, 30 min of normothermic aortic cross-clamping) and group 3 (n = 18, aprikalim 100 μg/kg was administered 15 min before 30 min of normothermic aortic cross-clamping). Neurologic evaluation was performed according to the modified Tarlov scale. Six animals from each group were sacrificed at 24, 48 and 168 h postoperatively. The lumbar spinal cords were harvested and examined histologically. The motor neurons were counted and the histologic lesions were scored (0-3, 3: normal). Group 3 (aprikalim group) had better Tarlov scores compared to group 2 at all-time points (P < 0.025). The histologic changes were proportional to the Tarlov scores and group 3 had better functional outcome as compared to group 2 at 168 h (number of neurons: 21.2 ± 4.9 vs. 8.0 ± 2.7, P < 0.001 and histologic score: 1.67 ± 1.03 vs. 0.50 ± 0.55, P = 0.03). Although aprikalim exhibited improved effect on clinical and histologic neurologic outcome when compared to normothermic spinal cord ischemia, animals in group 3 had worse Tarlov score, reduced number of motor neurons and worse histologic score when compared to group 1 (sham operation) at 168 h (P = 0.003, P = 0.001 and P = 0.019 respectively). Aprikalim reduces the severity of spinal cord ischemic injury in a rabbit model of spinal cord ischemia. Copyright © 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

  19. Garlic capsule and selenium-vitamins ACE combination therapy modulate key antioxidant proteins and cellular adenosine triphosphate in lisinopril-induced lung damage in rats.

    PubMed

    Akintunde, Jacob K; Bolarin, Olakunle Enock; Akintunde, Daniel G

    2016-03-01

    Garlic capsule (GAR) and/or selenium- vitamin A, C, E (S-VACE) might be useful in the treatment of lung diseases. The present study evaluated the toxicity of lisinopril (LIS) in the lungs of male rats and the reversal effect of GAR and/or selenium-vitamins A, C, and E (S-VACE). Group I served as the control, whereas animals in groups II, III, IV, and V received 28 mg of LIS/kg body weight by gavage. Group III was co-treated with GAR at a therapeutic dosage of 250 mg/kg body weight per day. Group IV was co-treated with S-VACE at dosage of 500 mg/kg body weight per day. Lastly, group V was co-treated with GAR and S-VACE at dosages of 250 and 500 mg/kg body weight per day, respectively. The experiment lasted for 8 days (sub-acute exposure). Administration of therapeutic dose of LIS to male rats depleted enzymatic antioxidants (superoxide dismutase and catalase) and cellular adenosine triphosphate content with concomitant increase in lipid peroxidation. Histopathology examination showed damage to the epithelial cells of the airways. These effects were prevented by both single and combination treatment of GAR and S-VACE in male rats with LIS-induced lung toxicity. We therefore concluded that the combination of GAR and S-VACE can be a novel therapy for the management of lung diseases in humans.

  20. Adiabatic Pumping Mechanism for Ion Motive ATPases

    NASA Astrophysics Data System (ADS)

    Astumian, R. Dean

    2003-09-01

    An ion motive ATPase is a membrane protein that pumps ions across the membrane at the expense of the chemical energy of adenosine triphosphate (ATP) hydrolysis. Here we describe how an external electric field, by inducing transitions between several protein configurations, can also power this pump. The underlying mechanism may be very similar to that of a recently constructed adiabatic electron pump [

    M. Switkes et al., Science 283, 1905 (1999)
    ].

  1. The effect of experimental gastric dilatation-volvulus on adenosine triphosphate content and conductance of the canine gastric and jejunal mucosa.

    PubMed

    Peycke, Laura E; Hosgood, Giselle; Davidson, Jacqueline R; Tetens, Joanne; Taylor, H Wayne

    2005-07-01

    The objective of this study was to determine if experimental gastric dilatation volvulus (GDV) would decrease adenosine triphosphate (ATP) concentration and increase membrane conductance of the canine gastric and jejunal mucosa. Male dogs (n = 15) weighing between 20 and 30 kg were used. Dogs were randomly assigned to 1 of 3 equal groups: Group 1 was control, group 2 was GDV, and group 3 was ischemia. All dogs were anesthetized for 210 min. Group 1 had no manipulation. Group 2 had GDV experimentally induced for 120 min followed by decompression, derotation, and reperfusion for 90 min. Group 3 had GDV experimentally induced for 210 min. Gastric (fundus and pylorus) and jejunal tissue was taken at 0, 120, and 210 min from all of the dogs. Tissue was analyzed for ATP concentration, mucosal conductance, and microscopic changes. The ATP concentration in the fundus did not change significantly from baseline in group 2, but decreased significantly below baseline at 210 min in group 3. The ATP concentration in the jejunum decreased significantly below baseline in groups 2 and 3 at 120 min, remaining significantly decreased in group 3 but returning to baseline at 210 min in group 2. Mucosal conductance of the fundus did not change significantly in any dog. Mucosal conductance of the jejunum increased at 120 min in groups 2 and 3, and became significantly increased above baseline at 210 min. The jejunal mucosa showed more profound cellular changes than the gastric mucosa. The jejunum showed substantial decreases in ATP concentration with an increase in mucosal conductance, suggesting cell membrane dysfunction. Dogs sustaining a GDV are likely to have a change in the activity of mucosal cells in the jejunum, which may be important in the pathophysiology of GDV.

  2. The effect of experimental gastric dilatation-volvulus on adenosine triphosphate content and conductance of the canine gastric and jejunal mucosa

    PubMed Central

    2005-01-01

    Abstract The objective of this study was to determine if experimental gastric dilatation volvulus (GDV) would decrease adenosine triphosphate (ATP) concentration and increase membrane conductance of the canine gastric and jejunal mucosa. Male dogs (n = 15) weighing between 20 and 30 kg were used. Dogs were randomly assigned to 1 of 3 equal groups: Group 1 was control, group 2 was GDV, and group 3 was ischemia. All dogs were anesthetized for 210 min. Group 1 had no manipulation. Group 2 had GDV experimentally induced for 120 min followed by decompression, derotation, and reperfusion for 90 min. Group 3 had GDV experimentally induced for 210 min. Gastric (fundus and pylorus) and jejunal tissue was taken at 0, 120, and 210 min from all of the dogs. Tissue was analyzed for ATP concentration, mucosal conductance, and microscopic changes. The ATP concentration in the fundus did not change significantly from baseline in group 2, but decreased significantly below baseline at 210 min in group 3. The ATP concentration in the jejunum decreased significantly below baseline in groups 2 and 3 at 120 min, remaining significantly decreased in group 3 but returning to baseline at 210 min in group 2. Mucosal conductance of the fundus did not change significantly in any dog. Mucosal conductance of the jejunum increased at 120 min in groups 2 and 3, and became significantly increased above baseline at 210 min. The jejunal mucosa showed more profound cellular changes than the gastric mucosa. The jejunum showed substantial decreases in ATP concentration with an increase in mucosal conductance, suggesting cell membrane dysfunction. Dogs sustaining a GDV are likely to have a change in the activity of mucosal cells in the jejunum, which may be important in the pathophysiology of GDV. PMID:16187546

  3. The Effect of Vitrification and in vitro Culture on the Adenosine Triphosphate Content and Mitochondrial Distribution of Mouse Pre-Implantation Embryos

    PubMed Central

    Amoushahi, Mahboobeh; Salehnia, Mojdeh; HosseinKhani, Saman

    2013-01-01

    Background: The mitochondria are an important source of adenosine triphosphate (ATP) production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. Methods: The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. Results: The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively (P>0.05). The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups (P>0.05). Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Conclusions: Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown. PMID:23748889

  4. Adenosine-derived doped carbon dots: From an insight into effect of N/P co-doping on emission to highly sensitive picric acid sensing.

    PubMed

    Li, Na; Liu, Shi Gang; Fan, Yu Zhu; Ju, Yan Jun; Xiao, Na; Luo, Hong Qun; Li, Nian Bing

    2018-07-12

    The various synthetic routes of carbon dots (C-dots) feature a considerable step toward their potential use in chemical sensors and biotechnology. Herein, by coupling phosphorus and nitrogen element introduction, the adenosine-derived N/P co-doped C-dots with fluorescence enhancement were achieved. By separately employing adenosine, adenosine monophosphate, adenosine diphosphate, and adenosine-5'-triphosphate as precursors, the effect of N/P co-doping on the fluorescence emission is discussed in detail. The formed C-dots with adenosine monophosphate exhibited strong blue fluorescence with a high quantum yield of 33.81%. Then the C-dots were employed as a fluorescent probe and utilized to develop a fast, sensitive, and selective picric acid sensor. The fluorescence of C-dots can be quenched by picric acid immediately, giving rise to a picric acid determination down to 30 nM. The possible mechanism of fluorescence quenching was discussed, which was proved to be inner filter effect and static quenching. Moreover, this method has the potential to detect picric acid in environmental water samples. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2009-04-01

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

  6. An improved red blood cell additive solution maintains 2,3-diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage.

    PubMed

    Burger, Patrick; Korsten, Herbert; De Korte, Dirk; Rombout, Eva; Van Bruggen, Robin; Verhoeven, Arthur J

    2010-11-01

    Current additive solutions (ASs) for red blood cells (RBCs) do not maintain constant 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) levels during cold storage. We have previously shown that with a new AS called phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM), both 2,3-DPG and ATP could be maintained throughout storage for 35 days. In this study, the mechanism underlying the effect of PAGGGM on RBC storage was studied in more detail. By using double-erythrocytapheresis units (leukoreduced), a direct comparison could be made between the current AS saline-adenine-glucose-mannitol (SAGM) and the experimental solution PAGGGM. During cold storage, several in vitro characteristics were analyzed. In agreement with our previous findings with single RBCs, PAGGGM maintained 2,3-DPG and ATP levels for 35 days of cold storage. Furthermore, glucose consumption and lactate production were higher in PAGGGM units during the first 21 days of cold storage. Fructose-1,6-diphophate and dihydroxyacetone phosphate levels were also increased during the first 21 days of storage in PAGGGM units. These results indicate that it is likely that phosphofructokinase (PFK) activity is enhanced in PAGGGM units relative to SAGM units. After 21 days, PFK activity also decreases in PAGGGM units, but sufficient metabolic reserve in these units prevents depletion of 2,3-DPG and ATP. © 2010 American Association of Blood Banks.

  7. Effects of chlorogenic acid, caffeine and coffee on components of the purinergic system of streptozotocin-induced diabetic rats.

    PubMed

    Stefanello, Naiara; Schmatz, Roberta; Pereira, Luciane Belmonte; Cardoso, Andréia Machado; Passamonti, Sabina; Spanevello, Rosélia Maria; Thomé, Gustavo; de Oliveira, Giovanna Medeiros Tavares; Kist, Luiza Wilges; Bogo, Maurício Reis; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

    2016-12-01

    We evaluated the effect of chlorogenic acid (CGA), caffeine (CA) and coffee (CF) on components of the purinergic system from the cerebral cortex and platelets of streptozotocin-induced diabetic rats. Animals were divided into eight groups: control animals treated with (I) water (WT), (II) CGA (5 mg/kg), (III) CA (15 mg/kg) and (IV) CF (0.5 g/kg), and diabetic animals treated with (V) WT, (VI) CGA (5 mg/kg), (VII) CA (15 mg/kg) and (VIII) CF (0.5 g/kg). Our results showed an increase (173%) in adenosine monophosphate (AMP) hydrolysis in the cerebral cortex of diabetic rats. In addition, CF treatment increased adenosine diphosphate (ADP) and AMP hydrolysis in group VIII synaptosomes. Platelets showed an increase in ectonucleotidase activity in group V, and all treatments reduced the increase in adenosine triphosphate and ADP hydrolysis. Furthermore, there was an increase in platelet aggregation of 72% in the diabetic rats, and CGA and CF treatment reduced platelet aggregation by nearly 60% when compared to diabetic rats. In this context, we can suggest that CGA and CF treatment should be considered a therapeutic and scientific target to be investigated in diseases associated with hyperglycemia. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2008-07-15

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

  9. Hypo-and hyperthyroidism affect the ATP, ADP and AMP hydrolysis in rat hippocampal and cortical slices.

    PubMed

    Bruno, Alessandra Nejar; Diniz, Gabriela Placoná; Ricachenevsky, Felipe Klein; Pochmann, Daniela; Bonan, Carla Denise; Barreto-Chaves, Maria Luiza M; Sarkis, João José Freitas

    2005-05-01

    The presence of severe neurological symptoms in thyroid diseases has highlighted the importance of thyroid hormones in the normal functioning of the mature brain. Since, ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system (CNS), the ectonucleotidase cascade that hydrolyzes ATP to adenosine, is also involved in the control of brain functions. Thus, we investigated the influence of hyper-and hypothyroidism on the ATP, ADP and AMP hydrolysis in hippocampal and cortical slices from adult rats. Hyperthyroidism was induced by daily injections of l-thyroxine (T4) 25 microg/100 g body weight, for 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Hypothyroid rats were hormonally replaced by daily injections of T4 (5 microg/100 g body weight, i.p.) for 5 days. Hyperthyroidism significantly inhibited the ATP, ADP and AMP hydrolysis in hippocampal slices. In brain cortical slices, hyperthyroidism inhibited the AMP hydrolysis. In contrast, hypothyroidism increased the ATP, ADP and AMP hydrolysis in both hippocampal and cortical slices and these effects were reverted by T4 replacement. Furthermore, hypothyroidism increased the expression of NTPDase1 and 5'-nucleotidase, whereas hyperthyroidism decreased the expression of 5'-nucleotidase in hippocampus of adult rats. These findings demonstrate that thyroid disorders may influence the enzymes involved in the complete degradation of ATP to adenosine and possibly affects the responses mediated by adenine nucleotides in the CNS of adult rats.

  10. Alkaline phosphatase protects against renal inflammation through dephosphorylation of lipopolysaccharide and adenosine triphosphate.

    PubMed

    Peters, E; Geraci, S; Heemskerk, S; Wilmer, M J; Bilos, A; Kraenzlin, B; Gretz, N; Pickkers, P; Masereeuw, R

    2015-10-01

    Recently, two phase-II trials demonstrated improved renal function in critically ill patients with sepsis-associated acute kidney injury treated with the enzyme alkaline phosphatase. Here, we elucidated the dual active effect on renal protection of alkaline phosphatase. The effect of human recombinant alkaline phosphatase (recAP) on LPS-induced renal injury was studied in Sprague-Dawley rats. Renal function was assessed by transcutaneous measurement of FITC-sinistrin elimination in freely moving, awake rats. The mechanism of action of recAP was further investigated in vitro using conditionally immortalized human proximal tubular epithelial cells (ciPTEC). In vivo, LPS administration significantly prolonged FITC-sinistrin half-life and increased fractional urea excretion, which was prevented by recAP co-administration. Moreover, recAP prevented LPS-induced increase in proximal tubule injury marker, kidney injury molecule-1 expression and excretion. In vitro, LPS-induced production of TNF-α, IL-6 and IL-8 was significantly attenuated by recAP. This effect was linked to dephosphorylation, as enzymatically inactive recAP had no effect on LPS-induced cytokine production. RecAP-mediated protection resulted in increased adenosine levels through dephosphorylation of LPS-induced extracellular ADP and ATP. Also, recAP attenuated LPS-induced increased expression of adenosine A2A receptor. However, the A2A receptor antagonist ZM-241385 did not diminish the effects of recAP. These results indicate that the ability of recAP to reduce renal inflammation may account for the beneficial effect observed in septic acute kidney injury patients, and that dephosphorylation of ATP and LPS are responsible for this protective effect. © 2015 The British Pharmacological Society.

  11. In vitro release of adenosine triphosphate from the urothelium of human bladders with detrusor overactivity, both neurogenic and idiopathic.

    PubMed

    Kumar, Vivek; Chapple, Christopher R; Rosario, Derek; Tophill, Paul R; Chess-Williams, Russell

    2010-06-01

    There is increased evidence to suggest a role for nonadrenergic-noncholinergic neurotransmission in the pathogenesis of bladder dysfunction. In this set of experiments, we have assessed the contribution of the urothelium to purinergic activity by quantifying the amount of adenosine triphosphate (ATP) released from the urothelium of patients with idiopathic detrusor overactivity (IDO) and with neurogenic detrusor overactivity (NDO) and comparing these releases to those of controls. Bladder tissue with urodynamically and clinically proven NDO (n=8) and IDO (n=8) were included in this study. The carefully dissected urothelium was stimulated by mechanically stretching as well as electrically stimulating and the ATP; thus, release was quantified. We used a Lucy Anthos 1 luminometre (Anthos Labtec Instruments GmBH, Wals, Austria) to perform the assay. The results were analysed using Stingray software (Dazdaq Ltd, Brighton, UK). Both mechanical stretch and electric field stimulation (EFS) led to increased ATP release in both sets of tissues with overactivity compared to the controls; this rise was even more significant for the IDO urothelium (2416.7±479.8 pmol/g [p<0.005]) than for the NDO urothelium (133.1±22.4 pmol/g [p<0.01]); values for the controls were 77.6±16.2 pmol/g. ATP release following mechanical stretch was more sensitive to tetrodotoxin in bladders with NDO compared to those with IDO as well as to the controls, with ATP levels falling from 233.5±20.7 pmol/g to 107.2±11.6 pmol/g, expressed as percentage of basal levels (p<0.002). The experiments were performed in vitro, and the female patients were a mix of peri- and postmenopausal states. These experiments suggested a significant rise in ATP release from the urothelium of bladders with NDO as well as those with IDO in comparison to controls. Most of the ATP released from bladders with NDO is primarily from neuronal sources. Copyright © 2009 European Association of Urology. Published by Elsevier B

  12. Secreted adenosine triphosphate from Aggregatibacter actinomycetemcomitans triggers chemokine response.

    PubMed

    Ding, Q; Quah, S Y; Tan, K S

    2016-10-01

    Extracellular ATP (eATP) is an important intercellular signaling molecule secreted by activated immune cells or released by damaged cells. In mammalian cells, a rapid increase of ATP concentration in the extracellular space sends a danger signal, which alerts the immune system of an impending danger, resulting in recruitment and priming of phagocytes. Recent studies show that bacteria also release ATP into the extracellular milieu, suggesting a potential role for eATP in host-microbe interactions. It is currently unknown if any oral bacteria release eATP. As eATP triggers and amplifies innate immunity and inflammation, we hypothesized that eATP secreted from periodontal bacteria may contribute to inflammation in periodontitis. The aims of this study were to determine if periodontal bacteria secrete ATP, and to determine the function of bacterially derived eATP as an inducer of inflammation. Our results showed that Aggregatibacter actinomycetemcomitans, but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum, secreted ATP into the culture supernatant. Exposure of periodontal fibroblasts to filter sterilized culture supernatant of A. actinomycetemcomitans induced chemokine expression in an eATP-dependent manner. This occurred independently of cyclic adenosine monophosphate and phospholipase C, suggesting that ionotrophic P2X receptor is involved in sensing of bacterial eATP. Silencing of P2X7 receptor in periodontal fibroblasts led to a significant reduction in bacterial eATP-induced chemokine response. Furthermore, bacterial eATP served as a potent chemoattractant for neutrophils and monocytes. Collectively, our findings provide evidence for secreted ATP of A. actinomycetemcomitans as a novel virulence factor contributing to inflammation during periodontal disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Variability in platelet dense granule adenosine triphosphate release findings amongst patients tested multiple times as part of an assessment for a bleeding disorder.

    PubMed

    Badin, M S; Graf, L; Iyer, J K; Moffat, K A; Seecharan, J L; Hayward, C P M

    2016-12-01

    Lumi-aggregometry quantification of platelet dense granule adenosine triphosphate (ATP) release is commonly used for diagnosing platelet function disorders. As the test findings show considerable variability for healthy controls, we postulated that patient findings might also be variable and investigated patients who were assessed for dense granule ATP release defects more than once. Analyses were performed on prospectively collected data for first and second tests for subjects tested for dense granule ATP release defects more than once by the Hamilton Regional Laboratory Program (HRLMP) between January 2007 and June 2013 (cohort I). Similar analyses were performed for subjects who were recruited to a platelet disorder study (cohort II) and were assessed for ATP release defects more than once before October 2015. A total of 150 unique subjects had multiple ATP release tests. Results with individual agonists were variable for many subjects. While normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), impaired release with multiple agonists was confirmed in only some subjects (cohort I: 34%; cohort II: 54%). Inconsistent findings were common (cohort I: 36%; cohort II: 39%). ISTH bleeding scores showed no relationship to the test findings. The finding of impaired ATP release with 2 or more agonists on both tests was not associated with an increased likelihood of a definite bleeding disorder. The variability in platelet dense granule ATP release findings amongst patients assessed for diagnostic purposes suggests that the test has limited value for diagnosing platelet disorders. © 2016 John Wiley & Sons Ltd.

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

    PubMed Central

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

    2012-01-01

    β-D-3′-Azido-2′,3′-dideoxyguanosine (3′-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3′-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3′-azido-ddG in primary cells. To gain insight into their structure–activity–resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3′-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3′-azido-2,6-diaminopurine >3′-azido-6-chloropurine; 3′-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure–activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1. PMID:21914723

  15. Simultaneous measurement of adenosine triphosphate release and aggregation potentiates human platelet aggregation responses for some subjects, including persons with Quebec platelet disorder.

    PubMed

    Hayward, C P M; Moffat, K A; Castilloux, J-F; Liu, Y; Seecharan, J; Tasneem, S; Carlino, S; Cormier, A; Rivard, G E

    2012-04-01

    Platelet aggregometry and dense granule adenosine triphosphate (ATP) release assays are helpful to diagnose platelet disorders. Some laboratories simultaneously measure aggregation and ATP release using Chronolume® a commercial reagent containing D-luciferin, firefly luciferase and magnesium. Chronolume® can potentiate sub-maximal aggregation responses, normalising canine platelet disorder findings. We investigated if Chronolume® potentiates human platelet aggregation responses after observing discrepancies suspicious of potentiation. Among patients simultaneously tested by light transmission aggregometry (LTA) on two instruments, 18/43 (42%), including 14/24 (58%) with platelet disorders, showed full secondary aggregation with one or more agonists only in tests with Chronolume®. As subjects with Quebec platelet disorder (QPD) did not show the expected absent secondary aggregation responses to epinephrine in tests with Chronolume®, the reason for the discrepancy was investigated using samples from 10 QPD subjects. Like sub-threshold ADP (0.75 μM), Chronolume® significantly increased QPD LTA responses to epinephrine (p<0.0001) and it increased both initial and secondary aggregation responses, leading to dense granule release. This potentiation was not restricted to QPD and it was mimicked adding 1-2 mM magnesium, but not D-luciferin or firefly luciferase, to LTA assays. Chronolume® potentiated the ADP aggregation responses of QPD subjects with a reduced response. Furthermore, it increased whole blood aggregation responses of healthy control samples to multiple agonists, tested at concentrations used for the diagnosis of platelet disorders (p values <0.05). Laboratories should be aware that measuring ATP release with Chronolume® can potentiate LTA and whole blood aggregation responses, which alters findings for some human platelet disorders, including QPD.

  16. Mechanical stress-induced interleukin-1beta expression through adenosine triphosphate/P2X7 receptor activation in human periodontal ligament cells.

    PubMed

    Kanjanamekanant, K; Luckprom, P; Pavasant, P

    2013-04-01

    Mechanical stress is an important factor in maintaining homeostasis of the periodontium. Interleukin-1beta (IL-1β) and adenosine triphosphate (ATP) are considered potent inflammatory mediators. In macrophages, ATP-activated P2X7 receptor is involved in IL-1β processing and release. Our previous works demonstrated mechanical stress-induced expression of osteopontin and RANKL through the ATP/P2Y1 receptor in human periodontal ligament (HPDL) cells. This study was designed to examine the effect of mechanical stress on IL-1β expression in HPDL cells, as well as the mechanism and involvement of ATP and the P2 purinergic receptor. Cultured HPDL cells were treated with continuous compressive loading. IL-1β expression was analyzed at both mRNA and protein levels, using RT-PCR and ELISA, respectively. Cell viability was examined using the MTT assay. ATP was also used to stimulate HPDL cells. Inhibitors, antagonists and the small interfering RNA (siRNA) technique were used to investigate the role of ATP and the specific P2 subtypes responsible for IL-1β induction along with the intracellular mechanism. Mechanical stress could up-regulate IL-1β expression through the release of ATP in HPDL cells. ATP alone was also capable of increasing IL-1β expression. The induction of IL-1β was markedly inhibited by inhibitors and by siRNA targeting the P2X7 receptor. ATP-stimulated IL-1β expression was also diminished by intracellular calcium inhibitors. Our work clearly indicates the capability of HPDL cells to respond directly to mechanical stimulation. The results signified the important roles of ATP/P2 purinergic receptors, as well as intracellular calcium signaling, in mechanical stress-induced inflammation via up-regulation of the proinflammatory cytokine, IL-1β, in HPDL cells. © 2012 John Wiley & Sons A/S.

  17. Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A₂A receptor levels in rats subjected to neuroinflammation.

    PubMed

    Smith, Mark D; Bhatt, Dhaval P; Geiger, Jonathan D; Rosenberger, Thad A

    2014-06-04

    Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic

  18. 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

  19. Adenosine triphosphate postconditioning is associated with better preserved global and regional cardiac function during myocardial ischemia and reperfusion: a speckle tracking imaging-based echocardiologic study.

    PubMed

    Ren, Min; Liu, Yujie; Zhao, Huiya; Dong, Shixia; Jiang, Zhonghui; Li, Keting; Tian, Jiawei

    2016-10-01

    Effects of ischemic postconditioning (IPostC) and adenosine triphosphate (ATP)-mediated pharmacologic postconditioning (ATP-PPostC) on cardiac function were evaluated by speckle tracking imaging (STI)-based echocardiography. A myocardial I/R model was induced in rabbits by reversible ligation of the left ventricular branch of coronary artery. Rabbits were randomized into three groups: ischemia and reperfusion (IR) (no further intervention), IPostC, and ATP-PPostC groups. Cardiac function was evaluated by conventional and STI-based echocardiography. Myocardial necrosis, apoptosis, and myocardial mRNAs of apoptosis-related proteins (Bcl-2 and Bax) were evaluated. Speckle tracking imaging (STI)-based echocardiography revealed that IPostC and ATP-PPostC were associated with better preserved global and regional cardiac function, as indicated by significantly increased GLSrsys, GLSrd, GLSsys, SrLsys, SrLd, and SLsys in both groups (all P<.5). Subsequent pathologic studies indicate that the percentage of necrotic myocardium and permillage of apoptotic cells were significantly lower in the IPostC and ATP-PPostC groups than in the IR group (all P<.05). Moreover, both IPostC and ATP-PPostC were associated with increased Bcl-2 mRNA levels and reduced Bax mRNA levels. IPostC and ATP-PPostC may exert cardioprotective functions by better preservation of cardiac function during the I/R process and at least partly via attenuation of myocardial apoptosis. © 2016 John Wiley & Sons Ltd.

  20. Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

    PubMed Central

    Chao, Julie; Weathersbee, Carolyn J.

    1974-01-01

    Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

  1. Davydov Solitons in Polypeptides

    NASA Astrophysics Data System (ADS)

    Scott, A. C.

    1985-08-01

    The experimental evidence for self-trapping of amide-I (CO stretching) vibrational energy in crystalline acetanilide (a model protein) is reviewed and related to A. S. Davydov's theory of solitons as a mechanism for energy storage and transport in protein. Particular attention is paid to the construction of quantum states that contain N amide-I vibrational quanta. It is noted that the `N = 2' state is almost exactly resonant with the free energy that is released upon hydrolysis of adenosine triphosphate.

  2. Adenosine monophosphate as a mediator of ATP effects at P1 purinoceptors

    PubMed Central

    Ross, Fiona M; Brodie, Martin J; Stone, Trevor W

    1998-01-01

    When perfused with a medium containing no added magnesium and 4-aminopyridine (4AP) (50 μM) hippocampal slices generated epileptiform bursts of an interictal nature. We have shown in a previous study that adenosine 5′-triphosphate (ATP) depressed epileptiform activity and that this effect was blocked by the adenosine A1 receptor antagonist cyclopentyltheophylline but was not affected by adenosine deaminase. This implied that ATP might act indirectly at P1 receptors or at a xanthine-sensitive P2 receptor. The aim of the present study was to investigate further the action of ATP on epileptiform activity.ATP can be metabolized by ecto-nucleotidases to adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP) and adenosine, respectively. Each of these metabolites can activate receptors in its own right: P2 receptors for ADP and P1 receptors for AMP and adenosine.We now show that both AMP and ATP (50 μM) significantly decrease epileptiform discharge rate in a rapid and reversible manner. 5′Adenylic acid deaminase (AMP deaminase, AMPase) (0.2 u ml−1), when perfused alone did not significantly alter the discharge rate over the 10 min superfusion period used for drug application. When perfused concurrently with AMP (50 μM), AMP deaminase prevented the depressant effect of AMP on discharge rate.AMP deaminase, at a concentration of 0.2 u ml−1 which annulled the effect of AMP (50 μM), prevented the inhibitory activity of ATP (50 μM). A higher concentration of ATP (200 μM) depressed the frequency of spontaneous bursts to approximately 30% control and this response was also prevented by AMP deaminase.Superfusion of the slices with 5′-nucleotidase also prevented the inhibitory activity of ATP on epileptiform discharges.The results suggest that AMP mediates the inhibitory effects of ATP on epileptiform activity, a conclusion which can explain the earlier finding that cyclopentyltheophylline but not adenosine deaminase inhibited the

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

    PubMed Central

    2013-01-01

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

  4. Design of molecular beacons as signaling probes for adenosine triphosphate detection in cancer cells based on chemiluminescence resonance energy transfer.

    PubMed

    Zhang, Shusheng; Yan, Yameng; Bi, Sai

    2009-11-01

    In the present study, binary and triplex DNA molecular beacons, as signaling probes based on a luminol-H(2)O(2)-horseradish peroxidase (HRP)-fluorescein chemiluminescence resonance energy transfer (CRET) system and structure-switching aptamers for highly sensitive detection of small molecules, are developed using adenosine triphosphate (ATP) as a model analyte to demonstrate the generality of the strategy. This CRET process occurs from donor luminol to acceptor fluorescein, which is oxidized by H(2)O(2) and catalyzed by HRP. DNA aptamer for ATP is first attached on the surface of magnetic nanoparticles (MNPs). The cDNA linker has an extension that hybridizes with two other DNAs (LumAuNP-DNA and F-DNA) or three other DNAs (HRP-DNA, LumAuNP-DNA, and F-DNA) to fabricate CRET-BMBP-MNP or CRET-TMBP-MNP conjugates that provide the CRET signals. Thus, in the absence of ATP, when the MNPs are removed from the solution, they also take with them the linker DNA and the CRET signal probes, and no CRET signal can be detected. However, when ATP is introduced, a competition for the ATP aptamer between ATP and the cDNA linker occurs. As a result, CRET-BMBP and CRET-TMBP are forced to dissociate from the MNP surface based on the structure switching of the aptamer. The CRET signals are proportional to the concentration of ATP. In order to accelerate the rate of the aptamer structure-switching process, an invader DNA is introduced into the proposed strategy. The present CRET system provides a low detection limit of 1.1 x 10(-7) and 3.2 x 10(-7) M for ATP detection by BMBP and TMBP, respectively, which also exhibits a good selectivity for ATP detection. Sample assays of ATP in K562 leukemia cells and 4T1 breast cancer cells confirm the reliability and practicality of the protocol, which reveal a good prospect of this platform for biological sample analysis.

  5. Origin and ablation of the adenosine triphosphate induced atrial fibrillation after circumferential pulmonary vein isolation: effects on procedural success rate.

    PubMed

    Zhang, Jinlin; Tang, Cheng; Zhang, Yonghua; Su, X I

    2014-04-01

    Adenosine triphosphate (ATP) has been used to provoke dormant pulmonary vein (PV) conduction after circumferential PV isolation (CPVI). However, there have been no systematic studies examining the incidence and the mechanism of ATP-induced atrial fibrillation (AF) following CPVI in paroxysmal AF. In this study, we explore the mechanism of ATP-induced AF and assess the feasibility of eliminating this response by additional radiofrequency (RF) ablation. A total of 300 consecutive patients with paroxysmal AF underwent CPVI. After all PVs were isolated, intravenous ATP (40 mg) was administered during an intravenous isoproterenol (ISP) infusion (5 μg/min). AF was reproducibly induced by ATP in 39 patients. Non-PV foci were confirmed and located in 29 of these patients at the onset of AF, including 27 foci in the superior vena cava (SVC), 1 focus in the crista terminalis, and 1 focus near the antrum of the PV. In all these cases, ATP-induced AF was eliminated after the non-PV foci were successfully ablated. For the other 10 patients, the foci triggering AF could not be confirmed or located due to the transient effect of ATP, thus no further ablation was performed. After a mean follow-up period of 18.7 ± 6.4 (8-24) months, the success rate in the ATP-induced AF group was not significantly different compared with the conventional treatment group who did not exhibit ATP-induced AF (76.9% vs 67.3%; P = 0.25). But in the subgroup of which the ATP-induced AF could be eliminated by additional RF ablation, the success rate was significantly higher than the non-ATP inducible group (86.2% vs 67.3%; P = 0.04). A large proportion of the ATP-induced AF post CPVI were initiated by rapid firing in the SVC. Eliminating this response by additional ablation may have an influence on clinical results of paroxysmal AF ablation. © 2014 Wiley Periodicals, Inc.

  6. Amplified Peroxidase-Like Activity in Iron Oxide Nanoparticles Using Adenosine Monophosphate: Application to Urinary Protein Sensing.

    PubMed

    Yang, Ya-Chun; Wang, Yen-Ting; Tseng, Wei-Lung

    2017-03-22

    Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe 3 O 4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe 3 O 4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H 2 O 2 ) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe 3 O 4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe 2+ /Fe 3+ sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H 2 O 2 . Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe 3 O 4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe 3 O 4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe 3 O 4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe 3 O 4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe 3 O 4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

  7. Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus.

    PubMed

    Kimura, Yoshio; Tanaka, Chihiro; Oka, Manami

    2018-07-01

    Myxococcus xanthus generates diadenosine tetraphosphates (Ap 4 A) and diadenosine pentaphosphates (Ap 5 A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap 4 A from ATP, Ap 5 A from ATP and adenosine tetraphosphate (Ap 4 ), and Ap 4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap 4 A and Ap 4 synthesis, 15 M. xanthus aminoacyl-tRNA synthetases (aaRSs), four acyl-CoA synthetases (Acys), three acetyl-CoA synthetases (Aces), phosphoglycerate kinase (Pgk), and adenylate kinase (Adk) were expressed in Escherichia coli and examined for Ap 4 A or Ap 4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap 4 A synthetase activity. AlaRS, SerRS, and LeuRS1 showed high ADP synthetase activity with ATP as a substrate in the presence of pyrophosphatase, and also demonstrated the ability to produce Ap 4 from ATP and triphosphate in the absence of pyrophosphatase. Ap 4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap 4 A, suggesting that these enzymes prefer triphosphate over ATP as a substrate in the second reaction. Some of the recombinant M. xanthus Acys and Aces also synthesized Ap 4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap 4 from ATP and 3-phosphoglycerate in the presence of Mg 2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap 4 synthesis in M. xanthus.

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

    PubMed

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

    2016-09-09

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

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

    DTIC Science & Technology

    1986-01-01

    Paddle and Burnstock (326), Williams and Forrester (463), Forrester and Williams (151) and Clemens and Forrester (82) provide evidence that hypoxia may...an ATp4 - receptor. Fed. Proc. 45:208, 1986. (abstr) 99. Dahlen , S.E. and Hedqvist, P. ATP, B,y-methylene ATP andN adenosine inhibit non-cholinergic...regulation of skeletal muscle blood low. Circ Res. 29:375-384, 1971. 117. Dodd, J., Jahr, C.E., Hamilton, P.N., Heath, M.J., Matthew , W.P., and Jessell, T.M

  10. Hydrolysis of aspartic acid phosphoramidate nucleotides: a comparative quantum chemical study.

    PubMed

    Michielssens, Servaas; Tien Trung, Nguyen; Froeyen, Matheus; Herdewijn, Piet; Tho Nguyen, Minh; Ceulemans, Arnout

    2009-09-07

    L-Aspartic acid has recently been found to be a good leaving group during HIV reverse transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. This showed that L-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis of several models for L-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic environment was mimicked by involving two Mg(2+) ions and some important active site residues in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier is lower than the comparable mechanism with methanol as a leaving group. The calculations on the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for L-aspartic-dAMP than for L-malic-dAMP and pyrophosphate.

  11. Determination of intracellular fludarabine triphosphate in human peripheral blood mononuclear cells by LC-MS/MS.

    PubMed

    Huang, Liusheng; Lizak, Patricia; Aweeka, Francesca; Long-Boyle, Janel

    2013-12-01

    Fludarabine is a nucleoside analog routinely used in conditioning regimens of pediatric allogeneic stem cell transplantation to promote stem cell engraftment. In children, it remains a challenge to accurately and precisely quantify the active intracellular triphosphate species of fludarabine in vivo, primarily due to limitations on blood volume and inadequate assay sensitivity. Here we report a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of fludarabine triphosphate in human peripheral blood mononuclear cells (PBMC). PBMC (∼5 million cells) were collected and lysed in 1mL 70% methanol containing 1.2mM tris buffer (pH 7.4). The lysate (80μL) was mixed with internal standard (2-chloro-adenosine triphosphate, 150ng/mL, 20μL) and injected onto an API5000 LC-MS/MS system. Separation was achieved on a hypercarb column (100mm×2.1mm, 3μm) eluted with 100mM ammonium acetate (pH 9.8) and acetonitrile in a gradient mode at a flow rate of 0.4mL/min. Multiple reactions monitoring (MRM) and electrospray ionization in negative mode (ESI(-)) were used for detection. The ion pairs 524.0/158.6 for the drug and 540.0/158.8 for the IS were selected for quantification and 524.0/425.7 used for confirmation. Retention time was 3.0 and 3.4min for fludarabine triphosphate and the IS, respectively. The concentration range for the calibration curve was 1.52-76nM. Our method is simple, fast, and has been successfully applied in a clinical dose-concentration study in children to quantify intracellular fludarabine in low volume clinical samples. The median concentration was 1.03 and 3.19pmole/million PBMC at trough and peak time points, respectively. Fludarabine triphosphate is degraded in water within hours but relatively stable in 70% methanol-tris (1.2mM, pH 7.4). One limitation is that the hypercarb column takes a longer time to equilibrate than conventional reverse phase columns, and peaks become broad and distorted if the column is not washed

  12. Fundamental Characteristics of AAA+ Protein Family Structure and Function

    PubMed Central

    2016-01-01

    Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins. PMID:27703410

  13. Fundamental Characteristics of AAA+ Protein Family Structure and Function.

    PubMed

    Miller, Justin M; Enemark, Eric J

    2016-01-01

    Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

  14. A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

    PubMed

    First, Eric A

    2015-08-15

    A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. ABC gene expression profiles have clinical importance and possibly form a new hallmark of cancer.

    PubMed

    Dvorak, Pavel; Pesta, Martin; Soucek, Pavel

    2017-05-01

    Adenosine triphosphate-binding cassette proteins constitute a large family of active transporters through extracellular and intracellular membranes. Increased drug efflux based on adenosine triphosphate-binding cassette protein activity is related to the development of cancer cell chemoresistance. Several articles have focused on adenosine triphosphate-binding cassette gene expression profiles (signatures), based on the expression of all 49 human adenosine triphosphate-binding cassette genes, in individual tumor types and reported connections to established clinicopathological features. The aim of this study was to test our theory about the existence of adenosine triphosphate-binding cassette gene expression profiles common to multiple types of tumors, which may modify tumor progression and provide clinically relevant information. Such general adenosine triphosphate-binding cassette profiles could constitute a new attribute of carcinogenesis. Our combined cohort consisted of tissues from 151 cancer patients-breast, colorectal, and pancreatic carcinomas. Standard protocols for RNA isolation and quantitative real-time polymerase chain reaction were followed. Gene expression data from individual tumor types as well as a merged tumor dataset were analyzed by bioinformatics tools. Several general adenosine triphosphate-binding cassette profiles, with differences in gene functions, were established and shown to have significant relations to clinicopathological features such as tumor size, histological grade, or clinical stage. Genes ABCC7, A3, A8, A12, and C8 prevailed among the most upregulated or downregulated ones. In conclusion, the results supported our theory about general adenosine triphosphate-binding cassette gene expression profiles and their importance for cancer on clinical as well as research levels. The presence of ABCC7 (official symbol CFTR) among the genes with key roles in the profiles supports the emerging evidence about its crucial role in various

  16. Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

    PubMed Central

    Hatanaka, M; Del Giudice, R; Long, C

    1975-01-01

    Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas. PMID:236559

  17. Ribavirin suppresses hepatic lipogenesis through inosine monophosphate dehydrogenase inhibition: Involvement of adenosine monophosphate-activated protein kinase-related kinases and retinoid X receptor α.

    PubMed

    Satoh, Shinya; Mori, Kyoko; Onomura, Daichi; Ueda, Youki; Dansako, Hiromichi; Honda, Masao; Kaneko, Shuichi; Ikeda, Masanori; Kato, Nobuyuki

    2017-08-01

    Ribavirin (RBV) has been widely used as an antiviral reagent, specifically for patients with chronic hepatitis C. We previously demonstrated that adenosine kinase, which monophosphorylates RBV into the metabolically active form, is a key determinant for RBV sensitivity against hepatitis C virus RNA replication. However, the precise mechanism of RBV action and whether RBV affects cellular metabolism remain unclear. Analysis of liver gene expression profiles obtained from patients with advanced chronic hepatitis C treated with the combination of pegylated interferon and RBV showed that the adenosine kinase expression level tends to be lower in patients who are overweight and significantly decreases with progression to advanced fibrosis stages. In our effort to investigate whether RBV affects cellular metabolism, we found that RBV treatment under clinically achievable concentrations suppressed lipogenesis in hepatic cells. In this process, guanosine triphosphate depletion through inosine monophosphate dehydrogenase inhibition by RBV and adenosine monophosphate-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4, were required. In addition, RBV treatment led to the down-regulation of retinoid X receptor α (RXRα), a key nuclear receptor in various metabolic processes, including lipogenesis. Moreover, we found that guanosine triphosphate depletion in cells induced the down-regulation of RXRα, which was mediated by microtubule affinity regulating kinase 4. Overexpression of RXRα attenuated the RBV action for suppression of lipogenic genes and intracellular neutral lipids, suggesting that down-regulation of RXRα was required for the suppression of lipogenesis in RBV action. Conclusion : We provide novel insights about RBV action in lipogenesis and its mechanisms involving inosine monophosphate dehydrogenase inhibition, adenosine monophosphate-activated protein kinase-related kinases, and down-regulation of RXRα. RBV may be a

  18. Davydov solitons in polypeptides

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Scott, A.

    1984-10-01

    The experimental evidence for self-trapping of amide-I (CO stretching) vibrational energy in crystalline acetanilide (a model protein) is reviewed and related to A. S. Davydov's theory of solitons as a mechanism for energy storage and transport in protein. Particular attention is paid to the construction of quantum states that contain N amide-I vibrational quanta. It is noted that the N = 2 state is almost exactly resonant with the free energy that is released upon hydrolysis of adenosine triphosphate. 30 references, 4 figures, 3 tables.

  19. Cardioprotective benefits of adenosine triphosphate-sensitive potassium channel opener diazoxide are lost with administration after the onset of stress in mouse and human myocytes.

    PubMed

    Janjua, M Burhan; Makepeace, Carol M; Anastacio, Melissa M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2014-10-01

    Adenosine triphosphate-sensitive (KATP) potassium channel opener diazoxide (DZX) maintains myocyte volume and contractility during stress via an unknown mechanism when administered at the onset of stress. This study was performed to investigate the cardioprotective potential of DZX when added after the onset of the stresses of hyperkalemic cardioplegia, metabolic inhibition, and hypo-osmotic stress. Isolated mouse ventricular and human atrial myocytes were exposed to control Tyrode's solution (TYR) for 10 to 20 minutes, test solution for 30 minutes (hypothermic hyperkalemic cardioplegia [CPG], CPG + 100uM diazoxide [CPG+DZX], metabolic inhibition [MI], MI+DZX, mild hypo-osmotic stress [0.9T], or 0.9T + DZX), with DZX added after 10 or 20 minutes of stress, followed by 20 minutes of re-exposure to TYR (±DZX). Myocyte volume (human + mouse) and contractility (mouse) were compared. Mouse and human myocytes demonstrated significant swelling during exposure to CPG, MI, and hypo-osmotic stress that was not prevented by DZX when administered either at 10 or 20 minutes after the onset of stress. Contractility after the stress of CPG in mouse myocytes significantly declined when DZX was administered 20 minutes after the onset of stress (p < 0.05 vs TYR). Contractility after hypo-osmotic stress in mouse myocytes was not altered by the addition of DZX. To maintain myocyte volume homeostasis and contractility during stress (hyperkalemic cardioplegia, metabolic inhibition, and hypo-osmotic stress), KATP channel opener diazoxide requires administration at the onset of stress in this isolated myocyte model. These data have potential implications for any future clinical application of diazoxide. Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

  20. Adenosine triphosphate stress myocardial perfusion imaging for risk stratification of patients aged 70 years and older with suspected coronary artery disease.

    PubMed

    Yao, Zhiming; Zhu, Hui; Li, Wenchan; Chen, Congxia; Wang, Hua; Shi, Lei; Zhang, Wenjie

    2017-04-01

    We investigated the cardiac risk stratification value of adenosine triphosphate stress myocardial perfusion imaging (ATP-MPI) in patients aged 70 years and older with suspected coronary artery disease (CAD). We identified a series of 415 consecutive patients aged 70 years and older with suspected CAD, who had undergone ATP-MPI with 99m Tc-MIBI. The presence of a fixed and/or reversible perfusion defect was considered as an abnormal MPI. Follow-up was available in 399 patients (96.1%) over 3.45 ± 1.71 years after excluding 16 patients who underwent early coronary revascularization <60 days after MPI. The major adverse cardiac events (MACE), including cardiac death, nonfatal infarction, and late coronary revascularization, were recorded. One hundred twenty-five (31.3%) patients had abnormal MPI and the remaining had normal MPI. A multivariable analysis using Cox regression demonstrated that abnormal MPI was independently associated with MACE (hazard ratio 19.50 and 95% confidence interval 5.91-64.31, P value .000). The patients with SSS > 8 had significantly higher cumulative MACE rate than patients with SSS ≤ 8 had (37.8% vs 5.2%, respectively, P < .001). The Kaplan-Meier cumulative MACE-free survival in patients with abnormal MPI (57.0%) was significantly lower than that in patients with normal MPI (89.6%), P < .0001. Among patients with SSS > 8, the Kaplan-Meier cumulative MACE-free survival were 36.9% in patients ≥80 years old and 49.5% in patients 70-79 years old, respectively, P < .05. However, among patients with SSS ≤ 8, there was no difference between the Kaplan-Meier cumulative MACE-free survivals of these two age groups. ATP-MPI data are useful for the prediction of major adverse cardiac events in patients aged 70 years and older with suspected CAD.

  1. Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

    PubMed

    Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

    2010-07-01

    Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

  2. Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression and in situ enzyme activity.

    PubMed

    Villamonte, María Lina; Torrejón-Escribano, Benjamín; Rodríguez-Martínez, Aitor; Trapero, Carla; Vidal, August; Gómez de Aranda, Inmaculada; Sévigny, Jean; Matías-Guiu, Xavier; Martín-Satué, Mireia

    2018-03-01

    Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those taking place in oviducts, including contraction, beating of cilia, and maintenance of fluid composition that, in turn, influences sperm capacitation and hyperactivation, as well as oocyte and embryo nourishing. Ecto-nucleotidases are the enzymes that regulate extracellular ATP and adenosine levels, thus playing a role in reproduction. We have optimized a convenient method for characterizing ecto-nucleotidases that simultaneously localizes the protein and its associated enzyme activity in the same tissue slice and characterizes ecto-nucleotidases in human oviducts. The technique combines immunofluorescence and in situ histochemistry, allowing precise identification of ecto-nucleotidases at a subcellular level. In oviducts, remarkably, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, with the ability to hydrolyze ATP to AMP, are expressed in ciliated epithelial cells but with different subcellular localization. Ecto-5'nucleotidase/CD73 is also expressed apically in ciliated cells. CD73, together with alkaline phosphatase, also expressed apically in oviductal epithelium, complete the hydrolysis sequence by dephosphorylating AMP to adenosine. The concerted action of these enzymes would contribute to the local increase of adenosine concentration necessary for sperm capacitation. The use of this method would be an asset for testing new potential therapeutic drugs with inhibitory potential, which is of great interest presently in the field of oncology and in other clinical disciplines.

  3. A synthetic DNA motor that transports nanoparticles along carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Cha, Tae-Gon; Pan, Jing; Chen, Haorong; Salgado, Janette; Li, Xiang; Mao, Chengde; Choi, Jong Hyun

    2014-01-01

    Intracellular protein motors have evolved to perform specific tasks critical to the function of cells such as intracellular trafficking and cell division. Kinesin and dynein motors, for example, transport cargoes in living cells by walking along microtubules powered by adenosine triphosphate hydrolysis. These motors can make discrete 8 nm centre-of-mass steps and can travel over 1 µm by changing their conformations during the course of adenosine triphosphate binding, hydrolysis and product release. Inspired by such biological machines, synthetic analogues have been developed including self-assembled DNA walkers that can make stepwise movements on RNA/DNA substrates or can function as programmable assembly lines. Here, we show that motors based on RNA-cleaving DNA enzymes can transport nanoparticle cargoes--CdS nanocrystals in this case--along single-walled carbon nanotubes. Our motors extract chemical energy from RNA molecules decorated on the nanotubes and use that energy to fuel autonomous, processive walking through a series of conformational changes along the one-dimensional track. The walking is controllable and adapts to changes in the local environment, which allows us to remotely direct `go' and `stop' actions. The translocation of individual motors can be visualized in real time using the visible fluorescence of the cargo nanoparticle and the near-infared emission of the carbon-nanotube track. We observed unidirectional movements of the molecular motors over 3 µm with a translocation velocity on the order of 1 nm min-1 under our experimental conditions.

  4. REVIEWS OF TOPICAL PROBLEMS Molecular energy transducers of the living cell. Proton ATP synthase: a rotating molecular motor

    NASA Astrophysics Data System (ADS)

    Romanovsky, Yurii M.; Tikhonov, Alexander N.

    2010-12-01

    The free energy released upon the enzymatic hydrolysis of adenosine triphosphate (ATP) is the main source of energy for the functioning of the living cell and all multicellular organisms. The overwhelming majority of ATP molecules are formed by proton ATP synthases, which are the smallest macromolecular electric motors in Nature. This paper reviews the modern concepts of the molecular structure and functioning of the proton ATP synthase, and real-time biophysical experiments on the rotation of the 'rotor' of this macromolecular motor. Some mathematical models describing the operation of this nanosized macromolecular machine are described.

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

    PubMed Central

    2012-01-01

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

  6. 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

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

    EPA Science Inventory

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

  8. Reappraisal of the clinical implications of adenosine triphosphate in terms of the prediction of reconnection sites in cases with electrical isolation of the pulmonary veins.

    PubMed

    Okishige, Kaoru; Aoyagi, Hideshi; Ihara, Kensuke; Iwai, Shinsuke; Nakamura, Tomofumi; Yamashita, Mitsumi; Katoh, Nobutaka; Hasegawa, Tomoaki; Kawaguchi, Naohiko; Keida, Takehiko; Sasano, Tetsuo; Hirao, Kenzo

    2015-11-01

    Dormant conduction (DC) induced by intravenous adenosine triphosphate (ATP) after pulmonary vein (PV) isolation (PVI) could predict subsequent PV reconnection (RC) sites. This study aimed to investigate the relationship between the DC and RC sites during the long-term follow-up. Ninety-one consecutive patients (62 males; mean age, 62 ± 11 years) with symptomatic persistent (n = 18) or paroxysmal (n = 73) atrial fibrillation (AF) who underwent PVI were included in this study. After a successful PVI, we administered ATP to reveal the DC sites. In total, DC sites were observed in 46 (51%) patients, and all were left un-ablated after marking or tagging all of them using fluoroscopic images and a three-dimensional (3D) mapping system. After the follow-up period (14.8 ± 3.6 months), AF recurred in 29 (32%) patients, all of whom had a DC in the initial ablation session, and underwent redo sessions. We divided the DC sites into three groups; in group A, the RC sites differed from the DC sites, in group B, the RC sites were identical to the DC sites, and in group C, the RC sites involved both DC and other sites. As a result, 20 (69%), 3 (11.5%), and 6 (19.5%) patients belonged to groups A, B, and C, respectively. Statistical analyses comparing the agreement between DC and the RC sites yielded a weak relationship. DC sites implying RC sites had a weak agreement, and other options to predict RC sites will be required to improve the clinical benefit of CA of AF.

  9. Postischemic Treatment With Ethyl Pyruvate Prevents Adenosine Triphosphate Depletion, Ameliorates Inflammation, and Decreases Thrombosis in a Murine Model of Hind-Limb Ischemia and Reperfusion

    PubMed Central

    Crawford, Robert S.; Albadawi, Hassan; Atkins, Marvin D.; Jones, John J.; Conrad, Mark F.; Austen, William G.; Fink, Mitchell P.; Watkins, Michael T.

    2011-01-01

    Introduction Experiments were designed to investigate the effects of ethyl pyruvate (EP) in a murine model of hind-limb ischemia-reperfusion (IR) injury. Methods C57BL6 mice underwent 90 minutes of unilateral ischemia followed by 24 hours of reperfusion using two treatment protocols. For the preischemic treatment (pre-I) protocol, mice (n = 6) were given 300 mg/kg EP before ischemia, followed by 150 mg/kg of EP just before reperfusion and at 6 hours and 12 hours after reperfusion. In a postischemic treatment (post-I) protocol, mice (n = 7) were treated with 300 mg/kg EP at the end of the ischemic period, then 15 minutes later, and 2 hours after reperfusion and 150 mg/kg of EP at 4 hours, 6 hours, 10 hours, 16 hours, and 22 hours after reperfusion. Controls mice for both protocols were treated with lactated Ringers alone at time intervals identical to EP. Skeletal muscle levels of adenosine triphosphate (ATP), interleukin-1β, keratinocyte chemoattractant protein, and thrombin antithrombin-3 complex were measured. Skeletal muscle architectural integrity was assessed microscopically. Results ATP levels were higher in mice treated with EP compared with controls under the both treatment protocols (p = 0.02). Interleukin-1β, keratinocyte chemoattractant protein, thrombin antithrombin-3 complex (p < 0.05), and the percentage of injured fibers (p < 0.0001) were significantly decreased in treated versus control mice under the both protocols. Conclusion Muscle fiber injury and markers of tissue thrombosis and inflammation were reduced, and ATP was preserved with EP in pre-I and post-I protocols. Further investigation of the efficacy of EP to modulate IR injury in a larger animal model of IR injury is warranted. PMID:21217488

  10. Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

    PubMed

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-11-04

    Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

  11. Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

    PubMed Central

    2015-01-01

    Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

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

    PubMed

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

    2016-01-01

    Biomolecular immobilization and construction of the sensing platform are usually crucial for the successful development of a high-efficiency detection system. Herein we report on a novel and label-free signal-amplified aptasensing for sensitive electrochemical detection of small molecules (adenosine triphosphate, ATP, used in this case) by coupling with target-induced hybridization chain reaction (HCR) and the assembly of electroactive silver nanotags. The system mainly consisted of two alternating hairpin probes, a partial-pairing trigger-aptamer duplex DNA and a capture probe immobilized on the electrode. Upon target ATP introduction, the analyte attacked the aptamer and released the trigger DNA, which was captured by capture DNA immobilized on the electrode to form a newly partial-pairing double-stranded DNA. Thereafter, the exposed domain at trigger DNA could be utilized as the initator strand to open the hairpin probes in sequence, and propagated a chain reaction of hybridization events between two alternating hairpins to form a long nicked double-helix. The electrochemical signal derived from the assembled silver nanotags on the nicked double-helix. Under optimal conditions, the electrochemical aptasensor could exhibit a high sensitivity and a low detection limit, and allowed the detection of ATP at a concentration as low as 0.03 pM. Our design showed a high selectivity for target ATP against its analogs because of the high-specificity ATP-aptamer reaction, and its applicable for monitoring ATP in the spiking serum samples. Improtantly, the distinct advantages of the developed aptasensor make it hold a great potential for the development of simple and robust sensing strategies for the detection of other small molecules by controlling the apatmer sequence. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2017-08-01

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

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

    PubMed

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

    2017-04-01

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

  15. Oxygen Transport Changes in Canine Subjects Transfused with Stored Blood from Chronically Anemic Donors.

    DTIC Science & Technology

    diphosphoglycerate , and adenosine triphosphate occurred with storage in both sets. 2,3 diphosphoglycerate levels were slightly higher initially in...Adenosine triphosphate levels increased significantly and remained high 24 hr after transfusion. Red cell survival decreased with storage for both

  16. Adenosine and preeclampsia.

    PubMed

    Salsoso, Rocío; Farías, Marcelo; Gutiérrez, Jaime; Pardo, Fabián; Chiarello, Delia I; Toledo, Fernando; Leiva, Andrea; Mate, Alfonso; Vázquez, Carmen M; Sobrevia, Luis

    2017-06-01

    Adenosine is an endogenous nucleoside with pleiotropic effects in different physiological processes including circulation, renal blood flow, immune function, or glucose homeostasis. Changes in adenosine membrane transporters, adenosine receptors, and corresponding intracellular signalling network associate with development of pathologies of pregnancy, including preeclampsia. Preeclampsia is a cause of maternal and perinatal morbidity and mortality affecting 3-5% of pregnancies. Since the proposed mechanisms of preeclampsia development include adenosine-dependent biological effects, adenosine membrane transporters and receptors, and the associated signalling mechanisms might play a role in the pathophysiology of preeclampsia. Preeclampsia associates with increased adenosine concentration in the maternal blood and placental tissue, likely due to local hypoxia and ischemia (although not directly demonstrated), microthrombosis, increased catecholamine release, and platelet activation. In addition, abnormal expression and function of equilibrative nucleoside transporters is described in foetoplacental tissues from preeclampsia; however, the role of adenosine receptors in the aetiology of this disease is not well understood. Adenosine receptors activation may be related to abnormal trophoblast invasion, angiogenesis, and ischemia/reperfusion mechanisms in the placenta from preeclampsia. These mechanisms may explain only a low fraction of the associated abnormal transformation of spiral arteries in preeclampsia, triggering cellular stress and inflammatory mediators release from the placenta to the maternal circulation. Although increased adenosine concentration in preeclampsia may be a compensatory or adaptive mechanism favouring placental angiogenesis, a poor angiogenic state is found in preeclampsia. Thus, preeclampsia-associated complications might affect the cell response to adenosine due to altered expression and activity of adenosine receptors, membrane transporters

  17. Catch-slip bonds can be dispensable for motor force regulation during skeletal muscle contraction

    NASA Astrophysics Data System (ADS)

    Dong, Chenling; Chen, Bin

    2015-07-01

    It is intriguing how multiple molecular motors can perform coordinated and synchronous functions, which is essential in various cellular processes. Recent studies on skeletal muscle might have shed light on this issue, where rather precise motor force regulation was partly attributed to the specific stochastic features of a single attached myosin motor. Though attached motors can randomly detach from actin filaments either through an adenosine triphosphate (ATP) hydrolysis cycle or through "catch-slip bond" breaking, their respective contribution in motor force regulation has not been clarified. Here, through simulating a mechanical model of sarcomere with a coupled Monte Carlo method and finite element method, we find that the stochastic features of an ATP hydrolysis cycle can be sufficient while those of catch-slip bonds can be dispensable for motor force regulation.

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  19. The clinical relevance and prognostic significance of adenosine triphosphate ATP-binding cassette (ABCB5) and multidrug resistance (MDR1) genes expression in acute leukemia: an Egyptian study.

    PubMed

    Farawela, Hala M; Khorshied, Mervat M; Kassem, Neemat M; Kassem, Heba A; Zawam, Hamdy M

    2014-08-01

    Multidrug resistance (MDR1) represents a major obstacle in the chemotherapeutic treatment of acute leukemia (AL). Adenosine triphosphate ATP-binding cassette (ABCB5) and MDR1 genes are integral membrane proteins belonging to ATP-binding cassette transporters superfamily. The present work aimed to investigate the impact of ABCB5 and MDR1 genes expression on the response to chemotherapy in a cohort of Egyptian AL patients. The study included 90 patients: 53 AML cases and 37 ALL cases in addition to 20 healthy volunteers as controls. Quantitative assessment of MDR1 and ABCB5 genes expression was performed by quantitative real-time polymerase chain reaction. Additional prognostic molecular markers were determined as internal tandem duplications of the FLT3 gene (FLT3-ITD) and nucleophosmin gene mutation (NPM1) for AML cases, and mbcr-abl fusion transcript for B-ALL cases. In AML patients, ABCB5 and MDR1 expression levels did not differ significantly between de novo and relapsed cases and did not correlate with the overall survival or disease-free survival. AML patients were stratified according to the studied genetic markers, and complete remission rate was found to be more prominent in patients having low expression of MDR1 and ABCB5 genes together with mutated NPM1 gene. In ALL patients, ABCB5 gene expression level was significantly higher in relapsed cases and MDR1 gene expression was significantly higher in patients with resistant disease. In conclusion, the results obtained by the current study provide additional evidence of the role played by these genes as predictive factors for resistance of leukemic cells to chemotherapy and hence treatment outcome.

  20. Effects of adenosine 5'-monophosphate on epidermal turnover.

    PubMed

    Furukawa, Fukumi; Kanehara, Shoko; Harano, Fumiki; Shinohara, Shigeo; Kamimura, Junko; Kawabata, Shigekatsu; Igarashi, Sachiyo; Kawamura, Mitsuaki; Yamamoto, Yuki; Miyachi, Yoshiki

    2008-10-01

    The structure and function of the epidermis is maintained by cell renewal based on epidermal turnover. Epidermal turnover is delayed by aging, and it is thought that the delay of the epidermal turnover is a cause of aging alternation of skin. The epidermal turnover is related to the energy metabolism of epidermal basal cells. Adenosine 5'-triphosphate (ATP) is needed for cell renewal: cell division, and adenosine 5'-monophosphate (AMP) increases the amount of intracellular ATP. These findings suggest that AMP accelerates the epidermal turnover delayed by aging. This study investigated whether AMP and adenosine 5'-monophosphate disodium salt (AMP2Na) accelerates the epidermal turnover. An effect of AMP2Na on cell proliferation was examined by our counting of keratinocytes. An effect of AMP2Na on cell cycle was examined by our counting of basal cells in DNA synthetic period of hairless rats. The effects of AMP2Na (or AMP) on the epidermal turnover were examined by our measuring stratum corneum transit time by use of guinea pigs, and by our measuring stratum corneum surface area by use of hairless rats and in a clinical pharmacological study. The AMP2Na showed two different profiles on the proliferation of primary cultured keratinocytes. At a low concentration it induced cell growth, whereas at a high concentration it inhibited cell growth. The number of basal cells in the DNA synthetic period of AMP2Na was significantly higher than that of the vehicle in hairless rats. The stratum corneum transit time of AMP2Na was significantly shorter than that of the vehicle in guinea pigs. The corneocyte surface area of emulsion containing AMP2Na was significantly smaller than that of the vehicle in volunteers. We conclude that AMP promotes the cell proliferation and the cell cycle progression of epidermal basal cells and accelerates epidermal turnover safely. In addition, AMP is useful for skin rejuvenation in dermatology and aesthetic dermatology.

  1. Cerebrospinal fluid from subarachnoid haemorrhage patients causes excessive oxidative metabolism compared to vascular smooth muscle force generation.

    PubMed

    Pyne, G J; Cadoux-Hudson, T A; Clark, J F

    2001-01-01

    Cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients can stimulate vascular smooth muscle to generate force in vitro. CSF from SAH patients suffering from delayed ischaemic neurological deficits due to cerebral vasospasm can generate near maximal force in vitro and previous experiments have ascribed this generation of force to be a calcium mediated event. The intracellular calcium concentration has been demonstrated to rise during the vasospastic process. Calcium also stimulates oxidative metabolism as does adenosine diphosphate (ADP), the product of adenosine triphosphate (ATP) hydrolysis. Significant alteration in high energy metabolites such as ATP, ADP and phosphocreatine have also been demonstrated in various models of SAH mediated vasospasm. Vascular smooth muscle predominantly uses oxidative metabolism for force generation and reserves glycolytic metabolism for ion homeostasis. A decrease in oxidative metabolism during force generation would imply failing mitochondria and increased glycolytic high-energy phosphate supply. Increased oxidative metabolism would imply a decreased efficiency of the contractile apparatus or mitochondria. The aim of this study was to see if SAH CSF stimulation of porcine carotid artery oxidative metabolism was altered during force generation when compared with incremental calcium stimulation with potassium chloride depolarisation. CSF from patients (n = 10) who had subarachnoid haemorrhage stimulated force generation but with a significant 'right shift' in oxygen consumption. This 'right shift' is indicative of an increased energy cost for contractile work. These results suggest that vascular smooth muscle contractile apparatus, when stimulated by subarachnoid cerebrospinal fluid, is consuming excess adenosine triphosphate during force generation.

  2. A novel adenosine precursor 2',3'-cyclic adenosine monophosphate inhibits formation of post-surgical adhesions.

    PubMed

    Forman, Mervyn B; Gillespie, Delbert G; Cheng, Dongmei; Jackson, Edwin K

    2014-09-01

    Intraperitoneal adenosine reduces abdominal adhesions. However, because of the ultra-short half-life and low solubility of adenosine, optimal efficacy requires multiple dosing. Here, we compared the ability of potential adenosine prodrugs to inhibit post-surgical abdominal adhesions after a single intraperitoneal dose. Abdominal adhesions were induced in mice using an electric toothbrush to damage the cecum. Also, 20 μL of 95 % ethanol was applied to the cecum to cause chemically induced injury. After injury, mice received intraperitoneally either saline (n = 18) or near-solubility limit of adenosine (23 mmol/L; n = 12); 5'-adenosine monophosphate (75 mmol/L; n = 11); 3'-adenosine monophosphate (75 mmol/L; n = 12); 2'-adenosine monophosphate (75 mmol/L; n = 12); 3',5'-cyclic adenosine monophosphate (75 mmol/L; n = 19); or 2',3'-cyclic adenosine monophosphate (75 mmol/L; n = 20). After 2 weeks, adhesion formation was scored by an observer blinded to the treatments. In a second study, intraperitoneal adenosine levels were measured using tandem mass spectrometry for 3 h after instillation of 2',3'-cyclic adenosine monophosphate (75 mmol/L) into the abdomen. The order of efficacy for attenuating adhesion formation was: 2',3'-cyclic adenosine monophosphate > 3',5'-cyclic adenosine monophosphate ≈ adenosine > 5'-adenosine monophosphate ≈ 3'-adenosine monophosphate ≈ 2'-adenosine monophosphate. The groups were compared using a one-factor analysis of variance, and the overall p value for differences between groups was p < 0.000001. Intraperitoneal administration of 2',3'-cAMP yielded pharmacologically relevant levels of adenosine in the abdominal cavity for >3 h. Administration of 2',3'-cyclic adenosine monophosphate into the surgical field is a unique, convenient and effective method of preventing post-surgical adhesions by acting as an adenosine prodrug.

  3. Adenosine triphosphate-sensitive potassium channel blocking agent ameliorates, but the opening agent aggravates, ischemia/reperfusion-induced injury. Heart function studies in nonfibrillating isolated hearts.

    PubMed

    Tosaki, A; Hellegouarch, A

    1994-02-01

    This study was conducted to elucidate the role of the adenosine triphosphate (ATP)-sensitive potassium channel blocking agent glibenclamide and the opener cromakalim in the mechanism of reperfusion-induced injury. Recently, ATP-sensitive potassium channel openers have been proposed to reduce ischemia/reperfusion-induced injury, including arrhythmias and heart function. Thus, one might hypothesize that pharmacologic agents that enhance the loss of potassium ions in the myocardium through ATP-sensitive potassium channels would be arrhythmogenic, and agents that interfere with tissue potassium ion loss would be antiarrhythmic. Isolated "working" guinea pig hearts and phosphorus-31 nuclear magnetic resonance spectroscopy were used to study the recovery of myocardial function and phosphorus compounds after 30, 40 and 50 min of normothermic global ischemia followed by reperfusion in untreated control and glibenclamide- and cromakalim-treated groups. After 30 min of ischemia, 1, 3, 10 and 30 mumol/liter of glibenclamide dose-dependently reduced the incidence of reperfusion-induced ventricular fibrillation (total) from its control value of 92% to 75%, 33% (p < 0.05), 33% (p < 0.05) and 42% (p < 0.05), respectively. The incidence of ventricular tachycardia followed the same pattern. A reduction of arrhythmias was also observed after 40 and 50 min of ischemia followed by reperfusion in the glibenclamide-treated hearts. Cromakalim, at the same concentrations, did not reduce the incidence of reperfusion-induced arrhythmias. During reperfusion, glibenclamide (3 and 10 mumol/liter) improved the recovery of coronary blood flow, aortic flow, myocardial contractility and tissue ATP and creatine phosphate content, but cromakalim failed to ameliorate the recovery of postischemic myocardium compared with that in the drug-free control hearts. The preservation of myocardial potassium ions and phosphorus compounds by glibenclamide can improve the recovery of postischemic function, but

  4. A step into the RNA world: Conditional analysis of hydrogel formation of adenosine 5'-monophosphate induced by cyanuric acid.

    PubMed

    Yokosawa, Takumi; Enomoto, Ryota; Uchino, Sho; Hirasawa, Ito; Umehara, Takuya; Tamura, Koji

    2017-12-01

    Nucleotide polymerization occurs by the nucleophilic attack of 3'-oxygen of the 3'-terminal nucleotide on the α-phosphorus of the incoming nucleotide 5'-triphosphate. The π-stacking of mononucleotides is an important factor for prebiotic RNA polymerization in terms of attaining the proximity of two reacting moieties. Adenosine and adenosine 5'-monophosphate (AMP) are known to form hydrogel in the presence of cyanuric acid at neutral pH. However, we observed that other canonical ribonucleotides did not gel under the same condition. The π-stacking-induced hydrogel formation of AMP was destroyed at pH 2.0, suggesting that the protonation of N at position 1 of adenine abolished hydrogen bonding with the NH of cyanuric acid and resulted in the deformation of the hexad of adenine and cyanuric acid. A liquid-like gel was formed in the case of adenosine with cyanuric acid and boric acid, whereas AMP caused the formation of a solid gel, implying that the negative charge inherent to AMP prevented the formation of esters of boric acid with the cis-diols of ribose. Cyanuric acid-driven oligomerizations of AMP might have been the first crucial event in the foundation of the RNA world. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

    PubMed Central

    Naguib, Fardos N. M.; Rais, Reem H.; Al Safarjalani, Omar N.; el Kouni, Mahmoud H.

    2015-01-01

    Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ± 0.0002 mM, KATP = 0.05 ± 0.008 mM, and Vmax = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii. PMID:26112826

  6. Automatic instrument for chemical processing to detect microorganism in biological samples by measuring light reactions

    NASA Technical Reports Server (NTRS)

    Kelbaugh, B. N.; Picciolo, G. L.; Chappelle, E. W.; Colburn, M. E. (Inventor)

    1973-01-01

    An automated apparatus is reported for sequentially assaying urine samples for the presence of bacterial adenosine triphosphate (ATP) that comprises a rotary table which carries a plurality of sample containing vials and automatically dispenses fluid reagents into the vials preparatory to injecting a light producing luciferase-luciferin mixture into the samples. The device automatically measures the light produced in each urine sample by a bioluminescence reaction of the free bacterial adenosine triphosphate with the luciferase-luciferin mixture. The light measured is proportional to the concentration of bacterial adenosine triphosphate which, in turn, is proportional to the number of bacteria present in the respective urine sample.

  7. 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

  8. Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dowling, J.N.; Saha, A.K.; Glew, R.H.

    1987-05-01

    Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP3) was explored. When neutrophil phosphoinositides were labeled with TSP, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP2) over 2 h. Treatment of (TH)inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP2. Following fMLPmore » stimulation, the fractional reduction in PIP2 and the fractional increase in IP3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP3 was reduced by ACP pre-treatment. The reduction in IP3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP2 available for hydrolysis. However, some loss of IP3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP2, the prognitor of IP3, and by hydrolyzing IP3 itself.« less

  9. Adenosine and the Auditory System

    PubMed Central

    Vlajkovic, Srdjan M; Housley, Gary D; Thorne, Peter R

    2009-01-01

    Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A1, A2A, A2B, and A3. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A1 adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A1 and A2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea. PMID:20190966

  10. Mitochondrial deoxyribonucleoside triphosphate pools in thymidine kinase 2 deficiency.

    PubMed

    Saada, Ann; Ben-Shalom, Efrat; Zyslin, Rivka; Miller, Chaya; Mandel, Hanna; Elpeleg, Orly

    2003-10-24

    Deficiency of mitochondrial thymidine kinase (TK2) is associated with mitochondrial DNA (mtDNA) depletion and manifests by severe skeletal myopathy in infancy. In order to elucidate the pathophysiology of this condition, mitochondrial deoxyribonucleoside triphosphate (dNTP) pools were determined in patients' fibroblasts. Despite normal mtDNA content and cytochrome c oxidase (COX) activity, mitochondrial dNTP pools were imbalanced. Specifically, deoxythymidine triphosphate (dTTP) content was markedly decreased, resulting in reduced dTTP:deoxycytidine triphosphate ratio. These findings underline the importance of balanced mitochondrial dNTP pools for mtDNA synthesis and may serve as the basis for future therapeutic interventions.

  11. Autoradiography of P2x ATP receptors in the rat brain.

    PubMed Central

    Balcar, V. J.; Li, Y.; Killinger, S.; Bennett, M. R.

    1995-01-01

    1. Binding of a P2x receptor specific radioligand, [3H]-alpha,beta-methylene adenosine triphosphate ([3H]-alpha,beta-MeATP) to sections of rat brain was reversible and association/dissociation parameters indicated that it consisted of two saturable components. Non-specific binding was very low (< 7% at 10 nM ligand concentration). 2. The binding was completely inhibited by suramin (IC50 approximately 14-26 microM) but none of the ligands specific for P2y receptors such as 2-methylthio-adenosine triphosphate (2-methyl-S-ATP) and 2-chloro-adenosine triphosphate (2-C1-ATP) nor 2-methylthio-adenosine diphosphate (2-methyl-S-ADP) a ligand for the P2 receptor on blood platelets ('P2T' type) produced strong inhibitions except for P1,P4-di(adenosine-5')tetraphosphate (Ap4A). 3. Inhibitors of Na+,K(+)-dependent adenosine triphosphatase (ATPase) ouabain, P1-ligand adenosine and an inhibitor of transport of, respectively, adenosine and cyclic nucleotides, dilazep, had no effect. 4. The highest density of P2x binding sites was found to be in the cerebellar cortex but the binding sites were present in all major brain regions, especially in areas known to receive strong excitatory innervation. Images Figure 2 PMID:7670731

  12. Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release.

    PubMed

    Matos, R; Cordeiro, J M; Coelho, A; Ferreira, S; Silva, C; Igawa, Y; Cruz, F; Charrua, A

    2016-12-01

    Pathophysiological mechanisms of chronic visceral pain (CVP) are unknown. This study explores the association between the sympathetic system and bladder nociceptors activity by testing the effect of a prolonged adrenergic stimulation on transient receptor potential vanilloid 1 (TRPV1) activity and on urothelial adenosine triphosphate (ATP) release. Female Wistar rats received saline, phenylephrine (PHE), PHE + silodosin, PHE + naftopidil or PHE + prazosin. TRPV1 knockout and wild-type mice received saline or PHE. Visceral pain behaviour tests were performed before and after treatment. Cystometry was performed, during saline and capsaicin infusion. Fos immunoreactivity was assessed in L6 spinal cord segment. Human urothelial ATP release induced by mechanical and thermal stimulation was evaluated. Subcutaneous, but not intrathecal, PHE administration induced pain, which was reversed by silodosin, a selective alpha 1A adrenoceptor antagonist, but not by naftopidil, a relatively selective antagonist for alpha 1D adrenoceptor. Silodosin also reversed PHE-induced bladder hyperactivity and L6 spinal cord Fos expression. Thus, in subsequent experiments, only silodosin was used. Wild-type, but not TRPV1 knockout, mice exhibited phenylephrine-induced pain. Capsaicin induced a greater increase in voiding contractions in PHE-treated rats than in control animals, and silodosin reversed this effect. When treated with PHE, ATP release from human urothelial cells was enhanced either by mechanical stimulation or by lowering the thermal threshold of urothelial TRPV1, which becomes abnormally responsive at body temperature. This study suggests that the activation of peripheral alpha 1A adrenoceptors induces CVP, probably through its interaction with TRPV1 and ATP release. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  13. An Enzyme-Responsive "Turn-on" Fluorescence Polymeric Superamphiphile as a Potential Visualizable Phosphate Prodrug Delivery Vehicle.

    PubMed

    Yang, Xi; Shen, Shihong; Guo, Li; Tan, Jidong; Lei, Henxin; Wu, Jianghan; Zhao, Lei; Xiong, Tao; Wu, Youshen; Cheng, Yilong; Zhang, Yanfeng

    2018-06-01

    The development of inexpensive and highly efficient enzyme-responsive polymers has significantly contributed to targeted drug delivery systems. Here, a superamphiphile with a capability of fluorescent dissociation sensing is designed. It is constructed with negatively charged adenosine 5'-triphosphate (ATP) and negatively charged fluorescein diphosphate (FDP), which are used as fluorescence detection, and a cationic diblock copolymer methoxy-poly(ethylene glycol) 113 -b-poly(2-dimethyl-aminoethyl methacrylate) 70 . Upon addition of calf intestinal alkaline phosphatase, the superamphiphile disintegrates, presumably due to the enzymatic hydrolysis of ATP. This process is accompanied by an increase in the fluorescence emission intensity of fluorescein owing to the hydrolysis of FDP. The in vitro application of the superamphiphile is also proven. Thus, the "turn-on" fluorescence of the superamphiphile serves as a real-time module for detection of the disintegration of superamphiphile. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

    PubMed

    Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

    Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

  15. 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. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. AMP and adenosine are both ligands for adenosine 2B receptor signaling.

    PubMed

    Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

    2018-01-15

    Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Lynge, J; Juel, C; Hellsten, Y

    2001-01-01

    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. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km= 177 ± 36 μm and Vmax= 1.9 ± 0.2 nmol ml−1 s−1 (0.7 nmol (mg protein)−1 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). 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. 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. 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 skeletal muscle cells

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

    PubMed Central

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

    2012-01-01

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

  19. Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

    PubMed

    Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

    2017-08-01

    Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    ATP is a well-known energy supplier in cells. The idea to associate ATP to pharmaceutical formulations/biotechnological devices to promote cells activity by potentially modulating their microenvironment thus appears as an appealing novel approach. Since biomimetic nanocrystalline apatites have shown great promise for biomedical applications (bone regeneration, cells diagnostics/therapeutics, ⿦), thanks to a high surface reactivity and an intrinsically high biocompatibility, the present contribution was aimed at exploring ATP/apatite interactions. ATP adsorption on a synthetic carbonated nanocrystalline apatite preliminarily characterized (by XRD, FTIR, Raman, TG-DTA and SEM-EDX) was investigated in detail, pointing out a good agreement with Sips isothermal features. Adsorption characteristics were compared to those previously obtained on monophosphate nucleotides (AMP, CMP), unveiling some specificities. ATP was found to adsorb effectively onto biomimetic apatite: despite smaller values of the affinity constant KS and the exponential factor m, larger adsorbed amounts were reached for ATP as compared to AMP for any given concentration in solution. m < 1 suggests that the ATP/apatite adsorption process is mostly guided by direct surface bonding rather than through stabilizing intermolecular interactions. Although standard οGads ° was estimated to only ⿿4 kJ/mol, the large value of Nmax led to significantly negative effective οGads values down to ⿿33 kJ/mol, reflecting the spontaneous character of adsorption process. Vibrational spectroscopy data (FTIR and Raman) pointed out spectral modifications upon adsorption, confirming chemical-like interactions where both the triphosphate group of ATP and its nucleic base were involved. The present study is intended to serve as a basis for future research works involving ATP and apatite nanocrystals/nanoparticles in view of biomedical applications (e.g. bone tissue engineering, intracellular drug delivery, ⿦).

  1. Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weiler, Monica; Schmetzer, Helga; German Research Center for Environmental Health, Munich

    The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP andmore » GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.« less

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

    PubMed

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

    2017-02-01

    ABCB4 (MDR3) is an adenosine triphosphate (ATP)-binding cassette (ABC) transporter expressed at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene are responsible for several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ABCB4 missense variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Five disease-causing variations in these motifs have been identified in ABCB4 (G535D, G536R, S1076C, S1176L, and G1178S), three of which are homologous to the gating mutations of cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7; i.e., G551D, S1251N, and G1349D), that were previously shown to be function defective and corrected by ivacaftor (VX-770; Kalydeco), a clinically approved CFTR potentiator. Three-dimensional structural modeling predicted that all five ABCB4 variants would disrupt critical interactions in the binding of ATP and thereby impair ATP-induced nucleotide-binding domain dimerization and ABCB4 function. This prediction was confirmed by expression in cell models, which showed that the ABCB4 mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. As also hypothesized on the basis of molecular modeling, PC secretion activity of the mutants was rescued by the CFTR potentiator, ivacaftor (VX-770). Disease-causing variations in the ATP-binding sites of ABCB4 cause defects in PC secretion, which can be rescued by ivacaftor. These results provide the first experimental evidence that ivacaftor is a potential therapy for selected patients who harbor mutations in the ATP-binding sites of ABCB4. (Hepatology 2017;65:560-570). © 2016 by the American Association for the Study of Liver

  3. Iron from haemoglobin and haemin modulates nucleotide hydrolysis in Trichomonas vaginalis.

    PubMed

    Vieira, Patrícia de Brum; Silva, Nícolas Luiz Feijó; Kist, Luiza Wilges; Oliveira, Giovanna Medeiros Tavares de; Bogo, Maurício Reis; Carli, Geraldo Atillio de; Macedo, Alexandre José; Tasca, Tiana

    2015-04-01

    Extracellular ATP may act as a danger signalling molecule, inducing inflammation and immune responses in infection sites. The ectonucleotidases NTPDase and ecto-5'-nucleotidase are enzymes that modulate extracellular nucleotide levels; these enzymes have been previously characterised in Trichomonas vaginalis. Iron plays an important role in the complex trichomonal pathogenesis. Herein, the effects of iron on growth, nucleotide hydrolysis and NTPDase gene expression in T. vaginalis isolates from female and male patients were evaluated. Iron from different sources sustained T. vaginalis growth. Importantly, iron from haemoglobin (HB) and haemin (HM) enhanced NTPDase activity in isolates from female patients and conversely reduced the enzyme activity in isolates from male patients. Iron treatments could not alter the NTPDase transcript levels in T. vaginalis. Furthermore, our results reveal a distinct ATP, ADP and AMP hydrolysis profile between isolates from female and male patients influenced by iron from HB and HM. Our data indicate the participation of NTPDase and ecto-5'-nucleotidase in the establishment of trichomonas infection through ATP degradation and adenosine production influenced by iron.

  4. Removal of interfering nucleotides from brain extracts containing substance p. Effect of drugs on brain concentrations of substance p

    PubMed Central

    Laszlo, I.

    1963-01-01

    Several methods for removing interfering nucleotides, adenosine-5'-monophosphate and adenosine 5'-triphosphate from brain extracts have been studied. An enzymic method, using adenylic acid deaminase, has been found suitable. This deaminates adenosine monophosphate to 5'-inosinic acid, an inactive compound which does not influence the estimations of substance P. Owing to the adenosine triphosphatase content of the enzyme extract, adenosine triphosphate was also inactivated. For the estimation of adenosine monophosphate-deaminase activity, a simple colorimetric method is described which measures the ammonia liberated from adenosine monophosphate. Substance P in mouse brain extracts was estimated after treatment of the animals with various drugs, and after the enzymic removal of interfering nucleotides from the brain extracts. The drugs had no effect on the substance P content of mouse brain. The effect of drugs on the contractions of the guinea-pig ileum induced by substance P was also investigated, and the effect of drugs on the estimations of substance P in brain extracts is discussed. PMID:14066136

  5. Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.

    PubMed

    Brown, Katherine A; Harris, Derek F; Wilker, Molly B; Rasmussen, Andrew; Khadka, Nimesh; Hamby, Hayden; Keable, Stephen; Dukovic, Gordana; Peters, John W; Seefeldt, Lance C; King, Paul W

    2016-04-22

    The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3. Copyright © 2016, American Association for the Advancement of Science.

  6. Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brown, K. A.; Harris, D. F.; Wilker, M. B.

    The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3. The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complexmore » under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.« less

  7. Building blocks for the solution phase synthesis of oligonucleotides: regioselective hydrolysis of 3',5'-Di-O-levulinylnucleosides using an enzymatic approach.

    PubMed

    García, Javier; Fernández, Susana; Ferrero, Miguel; Sanghvi, Yogesh S; Gotor, Vicente

    2002-06-28

    A short and convenient synthesis of 3'- and 5'-O-levulinyl-2'-deoxynucleosides has been developed from the corresponding 3',5'-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis, avoiding several tedious chemical protection/deprotection steps. Thus, Candida antartica lipase B (CAL-B) was found to selectively hydrolyze the 5'-levulinate esters, furnishing 3'-O-levulinyl-2'-deoxynucleosides 3 in >80% isolated yields. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) and Candida antarctica lipase A (CAL-A) exhibit the opposite selectivity toward the hydrolysis at the 3'-position, affording 5'-O-levulinyl derivatives 4 in >70% yields. A similar hydrolysis procedure was successfully extended to the synthesis of 3'- and 5'-O-levulinyl-protected 2'-O-alkylribonucleosides 7 and 8. This work demonstrates for the first time application of commercial CAL-B and PSL-C toward regioselective hydrolysis of levulinyl esters with excellent selectivity and yields. It is noteworthy that protected cytidine and adenosine base derivatives were not adequate substrates for the enzymatic hydrolysis with CAL-B, whereas PSL-C was able to accommodate protected bases during selective hydrolysis. In addition, we report an improved synthesis of dilevulinyl esters using a polymer-bound carbodiimide as a replacement for dicyclohexylcarbodiimide (DCC), thus considerably simplifying the workup for esterification reactions.

  8. 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

  9. Characterization of a diadenosine tetraphosphate-receptor distinct from the ATP-purinoceptor in human tracheal gland cells.

    PubMed

    Saleh, A; Picher, M; Kammouni, W; Figarella, C; Merten, M D

    1999-11-12

    Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are not well known. ATP binds to P2 receptors and induced secretory leucocyte protease inhibitor (SLPI) secretion through formation of cyclic adenosine monophosphate and mobilization of intracellular [Ca(2+)]. Since diadenosine polyphosphates (ApnA) are also endogenous effectors of P2 receptors, we investigated their effects in a cell line (MM39) of human tracheal gland cells. Diadenosine tetraphosphates (Ap4A) induced significant stimulation (+50+/-12%) of SLPI secretion and to a similar extent to that of ATP (+65+/-10%). No significant effects were observed with diadenosine triphosphate (Ap3A), diadenosine pentaphosphate (Ap5A), ADP and 2-methylthio-adenosine triphosphate (2-MeS-ATP). Since Ap4A was weakly hydrolyzed (<2% of total), and the hydrolysis product was only inosine which is ineffective on cells, this Ap4A effect was not due to Ap4A hydrolysis in ATP and adenosine monophosphate (AMP). A mixture of Ap4A and ATP elicited only partial additive effects on SLPI secretion. ADP was shown to be a potent antagonist of ATP and Ap4A receptors, with IC(50)s of 0.8 and 2 microM, respectively. 2-MeS-ATP also showed antagonistic properties with IC(50)s of 20 and 30 microM for ATP- and Ap4A-receptors, respectively. Single cell intracellular calcium ([Ca(2+)](i)) measurements showed similar transient increases of [Ca(2+)](i) after ATP or Ap4A challenges. ATP desensitized the cell [Ca(2+)](i) responses to ATP and Ap4A, and Ap4A also desensitized the cell response to Ap4A. Nevertheless, Ap4A did not desensitize the cell [Ca(2+)](i) responses to ATP. In conclusion, both P2Y2-ATP-receptors and Ap4A-P2D-receptors seem to be present in tracheal gland cells. Ap4A may only bind to P2D-receptors whilst ATP may bind to both Ap4A- and ATP-receptors.

  10. Time-resolved photoelectron spectroscopy of adenosine and adenosine monophosphate photodeactivation dynamics in water microjets

    NASA Astrophysics Data System (ADS)

    Williams, Holly L.; Erickson, Blake A.; Neumark, Daniel M.

    2018-05-01

    The excited state relaxation dynamics of adenosine and adenosine monophosphate were studied at multiple excitation energies using femtosecond time-resolved photoelectron spectroscopy in a liquid water microjet. At pump energies of 4.69-4.97 eV, the lowest ππ* excited state, S1, was accessed and its decay dynamics were probed via ionization at 6.20 eV. By reversing the role of the pump and probe lasers, a higher-lying ππ* state was excited at 6.20 eV and its time-evolving photoelectron spectrum was monitored at probe energies of 4.69-4.97 eV. The S1 ππ* excited state was found to decay with a lifetime ranging from ˜210 to 250 fs in adenosine and ˜220 to 250 fs in adenosine monophosphate. This lifetime drops with increasing pump photon energy. Signal from the higher-lying ππ* excited state decayed on a time scale of ˜320 fs and was measureable only in adenosine monophosphate.

  11. Circadian variations of adenosine and of its metabolism. Could adenosine be a molecular oscillator for circadian rhythms?

    PubMed

    Chagoya de Sánchez, V

    1995-03-01

    The present review describes the biological implications of the periodic changes of adenosine concentrations in different tissues of the rat. Adenosine is a purine molecule that could have been formed in the prebiotic chemical evolution and has been preserved. The rhythmicity of this molecule, as well as its metabolism and even the presence of specific receptors, suggests a regulatory role in eukaryotic cells and in multicellular organisms. Adenosine may be considered a chemical messenger and its action could take place at the level of the same cell (autocrine), the same tissue (paracrine), or on separate organs (endocrine). Exploration of the circadian variations of adenosine was planned considering the liver as an important tissue for purine formation, the blood as a vehicle among tissues, and the brain as the possible acceptor for hepatic adenosine or its metabolites. The rats used in these studies were adapted to a dark-light cycle of 12 h with an unrestrained feeding and drinking schedule. The metabolic control of adenosine concentration in the different tissues studied through the 24-h cycle is related to the activity of adenosine-metabolizing enzyme: 5'-nucleotidase adenosine deaminase, adenosine kinase, and S-adenosylhomocysteine hydrolase. Some possibilities of the factors modulating the activity of these enzymes are commented upon. The multiphysiological action of adenosine could be mediated by several actions: (i) by interaction with extracellular and intracellular receptors and (ii) through its metabolism modulating the methylation pathway, possibly inducing physiological lipoperoxidation, or participating in the energetic homeostasis of the cell. The physiological meaning of the circadian variations of adenosine and its metabolism was focused on: maintenance of the energetic homeostasis of the tissues, modulation of membrane structure and function, regulation of fasting and feeding metabolic pattern, and its participation in the sleep-wake cycle. From

  12. A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases

    PubMed Central

    Guarnieri, Michael T.; Blagg, Brian S. J.

    2011-01-01

    Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening. PMID:21050069

  13. Downregulation of adenosine and adenosine 1 receptor contributes to neuropathic pain in resiniferatoxin neuropathy.

    PubMed

    Kan, Hung-Wei; Chang, Chin-Hong; Lin, Chih-Lung; Lee, Yi-Chen; Hsieh, Sung-Tsang; Hsieh, Yu-Lin

    2018-04-16

    The neurochemical effects of adenosine signaling in small-fiber neuropathy leading to neuropathic pain are yet to be explored in a direct manner. This study examined this system at the level of ligand (via the ectonucleotidase activity of prostatic acid phosphatase, PAP) and adenosine A1 receptors (A1Rs) in resiniferatoxin (RTX) neuropathy, a peripheral neurodegenerative disorder which specifically affects nociceptive nerves expressing transient receptor potential vanilloid type 1 (TRPV1). We conducted immunohistochemistry on dorsal root ganglion neurons (DRG), high-performance liquid chromatography (HPLC) for functional assays, and pharmacological interventions to alter PAP and A1Rs in mice with RTX neuropathy. In DRG of RTX neuropathy, PAP(+) neurons were reduced compared with vehicle-treated mice (P = 0.002) . Functionally, PAP ectonucleotidase activity was consequently reduced (i.e., the content of adenosine in DRG, P = 0.012). PAP(+) neuronal density was correlated with the degree of mechanical allodynia, which was reversed by intrathecal lumbar puncture (i.t.) injection of recombinant PAP with a dose-dependent effect. Furthermore, A1Rs were downregulated (P = 0.002), and this downregulation was colocalized with the TRPV1 receptor (31.0% ± 2.8%). Mechanical allodynia was attenuated in a dose-dependent response by i.t. injection of the A1R ligand, adenosine; however, no analgesia was evident when an exogenous adenosine was blocked by A1R antagonist. This study demonstrated dual mechanisms of neuropathic pain in TRPV1-induced neuropathy, involving a reduced adenosine system at both the ligand (adenosine) and receptor (A1Rs) levels.

  14. Adenosine and sleep

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yanik, G.M. Jr.

    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% andmore » 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.« less

  15. Adenosine 5'-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice.

    PubMed

    Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

    2014-01-09

    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.

  16. Active Calcium and Strontium Transport in Human Erythrocyte Ghosts

    PubMed Central

    Olson, Erik J.; Cazort, Ralph J.

    1969-01-01

    Both calcium and strontium could be transported actively from erythrocytes if adenosine triphosphate, guanosine triphosphate, or inosine triphosphate were included in the hypotonic medium used to infuse calcium or strontium into the cells. Acetyl phosphate and pyrophosphate were not energy sources for the transport of either ion. Neither calcium nor strontium transport was accompanied by magnesium exchange, and the addition of Mg++ to the reaction medium in a final concentration of 3.0 mmoles/liter did not promote the transport of either ion. In the absence of nucleotide triphosphates, the addition of 1.5 mmoles/liter of Sr++ to the reaction solution did not bring about active calcium transport and similarly 1.5 mmoles/liter of Ca++ did not bring about active strontium transport. The inclusion of 1.5 mmoles/liter of Ca++ or Sr++ in the reaction medium did not interfere with the transport of the other ion when the erythrocytes were infused with adenosine triphosphate. PMID:4304202

  17. 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

  18. A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

    PubMed

    Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu

    2018-02-05

    For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  19. Pain-relieving prospects for adenosine receptors and ectonucleotidases

    PubMed Central

    Zylka, Mark J.

    2010-01-01

    Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. In contrast, the efficacy of adenosine or adenosine receptor agonists at treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A1 receptor (A1R)-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A2A receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine. PMID:21236731

  20. Selective Phosphonylation of 5'-Adenosine Monophosphate (5'-AMP) via Pyrophosphite [PPi(III)].

    PubMed

    Kaye, Karl; Bryant, David E; Marriott, Katie E R; Ohara, Shohei; Fishwick, Colin W G; Kee, Terence P

    2016-11-01

    We describe here experiments which demonstrate the selective phospho-transfer from a plausibly prebiotic condensed phosphorus (P) salt, pyrophosphite [H 2 P 2 O 5 2- ; PPi(III)], to the phosphate group of 5'-adenosine mono phosphate (5'-AMP). We show further that this P-transfer process is accelerated both by divalent metal ions (M 2+ ) and by organic co-factors such as acetate (AcO - ). In this specific case of P-transfer from PPi(III) to 5'-AMP, we show a synergistic enhancement of transfer in the combined presence of M 2+ & AcO - . Isotopic labelling studies demonstrate that hydrolysis of the phosphonylated 5'-AMP, [P(III)P(V)-5'-AMP], proceeds via nuceophilic attack of water at the Pi(III) terminus.

  1. Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

    PubMed

    Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; Matsuoka, Isao; García-Sevilla, Jesús A

    2018-06-01

    Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A 1 receptor and G-protein was assessed by means of two [ 35 S]GTPγS binding assays, i.e., conventional filtration method and [ 35 S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A 1 receptor-mediated Gα i-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [ 35 S]GTPγS binding determined with conventional assay derives from functional activation of Gα i/o proteins (not restricted only to Gα i-3 ) coupled to adenosine A 1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [ 35 S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %E max values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A 1 receptor/G-protein interaction in postmortem human brain tissue.

  2. Mesenchymal Stem Cells Induce Expression of CD73 in Human Monocytes In Vitro and in a Swine Model of Myocardial Infarction In Vivo.

    PubMed

    Monguió-Tortajada, Marta; Roura, Santiago; Gálvez-Montón, Carolina; Franquesa, Marcella; Bayes-Genis, Antoni; Borràs, Francesc E

    2017-01-01

    The ectoenzymes CD39 and CD73 regulate the purinergic signaling through the hydrolysis of adenosine triphosphate (ATP)/ADP to AMP and to adenosine (Ado), respectively. This shifts the pro-inflammatory milieu induced by extracellular ATP to the anti-inflammatory regulation by Ado. Mesenchymal stem cells (MSCs) have potent immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype aiding tissue repair. In vitro , we observed that human cardiac adipose tissue-derived MSCs (cATMSCs) and umbilical cord MSCs similarly polarize monocytes toward a regulatory M2 phenotype, which maintained the expression of CD39 and induced expression of CD73 in a cell contact dependent fashion, correlating with increased functional activity. In addition, the local treatment with porcine cATMSCs using an engineered bioactive graft promoted the in vivo CD73 expression on host monocytes in a swine model of myocardial infarction. Our results suggest the upregulation of ectonucleotidases on MSC-conditioned monocytes as an effective mechanism to amplify the long-lasting immunomodulatory and healing effects of MSCs delivery.

  3. Adenosine kinase regulation of cardiomyocyte hypertrophy

    PubMed Central

    Fassett, John T.; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie

    2011-01-01

    There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKTSer473 phosphorylation but did attenuate sustained phosphorylation of RafSer338 (24–48 h), mTORSer2448 (24–48 h), p70S6kThr389 (2.5–48 h), and ERKThr202/Tyr204 (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6kThr389 phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERKThr202/Tyr204 and AKTSer473. Reduction of Raf-induced p70S6kThr389 phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of

  4. Adenosine kinase regulation of cardiomyocyte hypertrophy.

    PubMed

    Fassett, John T; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie; Bache, Robert J

    2011-05-01

    There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser⁴⁷³) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser³³⁸) (24-48 h), mTOR(Ser²⁴⁴⁸) (24-48 h), p70S6k(Thr³⁸⁹) (2.5-48 h), and ERK(Thr²⁰²/Tyr²⁰⁴) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser⁴⁷³). Reduction of Raf-induced p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte

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

    PubMed Central

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

    2007-01-01

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

  6. Neuroprotective effects of adenosine deaminase in the striatum

    PubMed Central

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

    2016-01-01

    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

  7. Ticagrelor Compared with Clopidogrel Increased Adenosine and Cyclic Adenosine Monophosphate Plasma Concentration in Acute Coronary Syndrome Patients.

    PubMed

    Li, Xiaoye; Wang, Qibing; Xue, Ying; Chen, Jiahui; Lv, Qianzhou

    2017-06-01

    Ticagrelor produces a more potent antiplatelet effect than clopidogrel and inhibits cellular uptake of adenosine, which is associated with several cardiovascular consequences. We aimed to explore the correlation between adenosine and cyclic adenosine monophosphate (cAMP) plasma concentration and antiplatelet effect by clopidogrel or ticagrelor in patients with acute coronary syndrome (ACS) receiving dual antiplatelet therapy (DAPT). We conducted a prospective, observational and single-centre cohort study enrolling 68 patients with non-ST-segment elevation ACS from January 2016 to May 2016. We monitored the inhibition of platelet aggregation (IPA) and assessed adenosine, adenosine deaminase (ADA) and cAMP plasma concentrations by immunoassay on admission and 48 hr after coronary angiography. The demographic and clinical data were collected by reviewing their medical records. The two groups exhibited similar baseline characteristics including adenosine, ADA and cAMP. The mean IPA in patients receiving ticagrelor was significantly higher than that in patients receiving clopidogrel (93.5% versus 67.2%; p = 0.000). Also, we observed that patients treated with ticagrelor had a significantly higher increase in levels of adenosine and cAMP compared with those treated with clopidogrel (1.04 (0.86; 1.41) versus 0.04 (-0.25; 0.26); p = 0.029 and 0.78 (-1.67; 1.81) versus 0.60 (-1.91; 4.60); p = 0.037, respectively). And there was a weak correlation between IPA and adenosine as well as cAMP plasma concentration (r = 0.390, p = 0.001 and r = 0.335, p = 0.005, respectively). Ticagrelor increased adenosine and cAMP plasma concentration compared with clopidogrel in patients with ACS. © 2017 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  8. 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

  9. Unexpected Effects of K+ and Adenosine Triphosphate on the Thermal Stability of Na+,K+-ATPase.

    PubMed

    Placenti, M Agueda; Kaufman, Sergio B; González Flecha, F Luis; González Lebrero, Rodolfo M

    2017-05-18

    Na + ,K + -ATPase is an integral membrane protein which couples ATP hydrolysis to the transport of three Na + out and two K + into the cell. The aim of this work is to characterize the effect of K + , ATP, and Mg 2+ (essential activator) on the Na + ,K + -ATPase thermal stability. Under all conditions tested, thermal inactivation of the enzyme is concomitant with a structural change involving the ATP binding site and membrane-associated regions. Both ligands exert a clear stabilizing effect due to both enthalpic and entropic contributions. Competition experiments between ATP and K + showed that, when ATP is present, the inactivation rate coefficient exhibits a biphasic dependence on K + concentration. At low [K + ], destabilization of the enzyme is observed, while stabilization occurred at larger cation concentrations. This is not expected for a simple competition between the enzyme and two ligands that individually protect the enzyme. A model that includes enzyme species with none, one, or two K + and/or one molecule of ATP bound explains the experimental data. We concluded that, despite both ligands stabilizing the enzyme, the species with one K + and one ATP simultaneously bound is unstable.

  10. Short-term hyperthyroidism modulates adenosine receptors and catalytic activity of adenylate cyclase in adipocytes.

    PubMed Central

    Rapiejko, P J; Malbon, C C

    1987-01-01

    The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate

  11. The examination of urine samples for pathogenic microbes by the luciferase assay for ATP. 1: The effect of the presence of fungi, fungal like bacteria and kidney cells in urine samples

    NASA Technical Reports Server (NTRS)

    Bush, V. N.

    1973-01-01

    A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.

  12. 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

  13. Ba-Wei-Die-Huang-Wan (Hachimi-jio-gan) can ameliorate cyclophosphamide-induced ongoing bladder overactivity and acidic adenosine triphosphate solution-induced hyperactivity on rats prestimulated bladder.

    PubMed

    Lee, Wei-Chia; Wu, Chia-Ching; Chuang, Yao-Chi; Tain, You-Lin; Chiang, Po-Hui

    2016-05-26

    Ba-Wei-Die-Huang-Wan (BWDHW) is the traditional Chinese medicine formula containing eight ingredients, namely Rehmannia glutinosa (Gaetn.) DC., root, steamed & dried; Cornus officinalis Siebold & Zucc., fructus, dried; Dioscorea oppositifolia L., root, dried; Alisma plantago-aquatica, subsp. orientale (Sam.) Sam., tuber, dried; Poria cocos (Fr.) Wolf., sclerotium, dried; Paeonia×suffruticosa Andrews, bark, dried; Cinnamomum cassia (Nees & T.Nees) J. Presl, bark, dried; Aconitum carmichaeli Debeaux, lateral root, dried & processed. It has been used for diabetes and urinary frequency treatments. We investigate effects of BWDHW on cyclophosphamide (CYP)-induced ongoing bladder overactivity and acidic adenosine triphosphate (ATP) solution-induced hyperactivity on rat's prestimulated bladder. Female Wistar rats were injected with intraperitoneal CYP (100mg/kg) or saline respectively. Rats were treated with BWDHW (90mg/kg/day) or vehicle for the next five days. After treatments animals were evaluated both in metabolic cage model and then by cystometry. Acidic ATP solution (5mM, pH 3.3) was instilled to provoke bladder hyperactivity. Bladder mucosa and muscle proteins were assessed by Western blotting. As compared to the controls, the CYP group showed significantly decreased mean cystometric intercontractile interval and increased micturition frequency, whereas the CYP/BWDWH group did not. The CYP group had significant protein overexpression in mucosal M2, M3, P2X2, and P2X3 receptors as well as detrusor M2 and M3 receptors. However, the CYP/BWDWH group had insignificant changes from controls. In the provoking test, the control/BWDHW and CYP/BWDHW groups were less affected by acidic ATP stimulation of intercontractile interval changes than the control group. Compared to the control group, the control/BWDHW group showed significantly lower mucosal P2X3 protein expression and the CYP group showed significant mucosal TRPV1 protein upregulation after the provoking test

  14. Caffeine and adenosine.

    PubMed

    Ribeiro, Joaquim A; Sebastião, Ana M

    2010-01-01

    Caffeine causes most of its biological effects via antagonizing all types of adenosine receptors (ARs): A1, A2A, A3, and A2B and, as does adenosine, exerts effects on neurons and glial cells of all brain areas. In consequence, caffeine, when acting as an AR antagonist, is doing the opposite of activation of adenosine receptors due to removal of endogenous adenosinergic tonus. Besides AR antagonism, xanthines, including caffeine, have other biological actions: they inhibit phosphodiesterases (PDEs) (e.g., PDE1, PDE4, PDE5), promote calcium release from intracellular stores, and interfere with GABA-A receptors. Caffeine, through antagonism of ARs, affects brain functions such as sleep, cognition, learning, and memory, and modifies brain dysfunctions and diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, Epilepsy, Pain/Migraine, Depression, Schizophrenia. In conclusion, targeting approaches that involve ARs will enhance the possibilities to correct brain dysfunctions, via the universally consumed substance that is caffeine.

  15. Difference in Energy Metabolism of Annulus Fibrosus and Nucleus Pulposus Cells of the Intervertebral Disc

    PubMed Central

    Salvatierra, Jessica Czamanski; Yuan, Tai Yi; Fernando, Hanan; Castillo, Andre; Gu, Wei Yong; Cheung, Herman S.; Huant, C.-Y. Charles

    2011-01-01

    Low back pain is associated with intervertebral disc degeneration. One of the main signs of degeneration is the inability to maintain extracellular matrix integrity. Extracellular matrix synthesis is closely related to production of adenosine triphosphate (i.e. energy) of the cells. The intervertebral disc is composed of two major anatomical regions: annulus fibrosus and nucleus pulposus, which are structurally and compositionally different, indicating that their cellular metabolisms may also be distinct. The objective of this study was to investigate energy metabolism of annulus fibrosus and nucleus pulposus cells with and without dynamic compression, and examine differences between the two cell types. Porcine annulus and nucleus tissues were harvested and enzymatically digested. Cells were isolated and embedded into agarose constructs. Dynamically loaded samples were subjected to a sinusoidal displacement at 2 Hz and 15% strain for 4 h. Energy metabolism of cells was analyzed by measuring adenosine triphosphate content and release, glucose consumption, and lactate/nitric oxide production. A comparison of those measurements between annulus and nucleus cells was conducted. Annulus and nucleus cells exhibited different metabolic pathways. Nucleus cells had higher adenosine triphosphate content with and without dynamic loading, while annulus cells had higher lactate production and glucose consumption. Compression increased adenosine triphosphate release from both cell types and increased energy production of annulus cells. Dynamic loading affected energy metabolism of intervertebral disc cells, with the effect being greater in annulus cells. PMID:21625336

  16. Adenosine and inflammation: what's new on the horizon?

    PubMed

    Antonioli, Luca; Csóka, Balázs; Fornai, Matteo; Colucci, Rocchina; Kókai, Endre; Blandizzi, Corrado; Haskó, György

    2014-08-01

    Adenosine contributes to the maintenance of tissue integrity by modulating the immune system. Encouraging results have emerged with adenosine receptor ligands for the management of several inflammatory conditions in preclinical and clinical settings. However, therapeutic applications of these drugs are sometimes complicated by the occurrence of serious adverse effects. The scientific community is making intensive efforts to design novel adenosine receptor ligands endowed with greater selectivity or to develop innovative compounds acting as allosteric receptor modulators. In parallel, research is focusing on novel pharmacological entities (designated as adenosine-regulating agents) that can increase, in a site- and event-specific manner, adenosine concentrations at the inflammatory site, thereby minimizing the adverse systemic effects of adenosine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors.

    PubMed

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32-35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR.

  18. REGULATION OF THE T-CELL RESPONSE BY CD39

    PubMed Central

    Takenaka, Maisa C.; Robson, Simon; Quintana, Francisco J.

    2016-01-01

    The ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, or CD39) catalyzes the phosphohydrolysis of extracellular adenosine triphosphate (eATP) and diphosphate (eADP) released under conditions of inflammatory stress and cell injury. CD39 generates adenosine monophosphate (AMP), which is in turn used by the ecto-5’-nucleotidase CD73 to synthesize adenosine. These ectonucleotidases have major impacts on the dynamic equilibrium of pro-inflammatory eATP and ADP nucleotides vs. immunosuppressive adenosine nucleosides. Indeed, CD39 plays a dominant role in the purinergic regulation of inflammation and the immune response because its expression is influenced by genetic and environmental factors. Here, we review the specific role of CD39 in the kinetic regulation of cellular immune responses in the evolution of disease. We focus on the effects of CD39 on T cells and explore potential clinical applications in autoimmunity, chronic infections and cancer. PMID:27236363

  19. CRYSTALLINE INORGANIC PYROPHOSPHATASE ISOLATED FROM BAKER'S YEAST

    PubMed Central

    Kunitz, M.

    1952-01-01

    Crystalline inorganic pyrophosphatase has been isolated from baker's yeast. The crystalline enzyme is a protein of the albumin type with an isoelectric point near pH 4.8. Its molecular weight is of the order of 100,000. It contains about 5 per cent tyrosine and 3.5 per cent tryptophane. It is most stable at pH 6.8. The new crystalline protein acts as a specific catalyst for the hydrolysis of inorganic pyrophosphate into orthophosphate ions. It does not catalyze the hydrolysis of the pyrophosphate radical of such organic esters as adenosine di- and triphosphate, or thiamine pyrophosphate. Crystalline pyrophosphatase requires the presence of Mg, Co, or Mn ions as activators. These ions are antagonized by calcium ions. Mg is also antagonized by Co or Mn ions. The rate of the enzymatic hydrolysis of inorganic pyrophosphate is proportional to the concentration of enzyme and is a function of pH, temperature, concentration of substrate, and concentration of activating ion. The approximate conditions for optimum rate are: 40°C. and pH 7.0 at a concentration of 3 to 4 x 10–3 M Na4P2O7 and an equivalent concentration of magnesium salt. The enzymatic hydrolysis of Na4P2O7 or K4P2O7 proceeds to completion and is irreversible under the conditions at which hydrolysis is occurring. Details are given of the method of isolation of the crystalline enzyme. PMID:14898026

  20. Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.

    PubMed

    Kellner, Julian N; Reinstein, Jochen; Meinhart, Anton

    2015-03-11

    RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an 'open' to a 'closed'-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    PubMed

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  2. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  3. Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

    PubMed Central

    Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

    2015-01-01

    Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vorontsov, Ivan I.; Minasov, George; Kiryukhina, Olga

    2012-06-19

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

  5. Is adenosine associated with sudden death in schizophrenia? A new framework linking the adenosine pathway to risk of sudden death.

    PubMed

    Gadelha, Ary; Zugman, André; Calzavara, Mariana Bendlin; de Mendonça Furtado, Remo Holanda; Scorza, Fulvio Alexandre; Bressan, Rodrigo Afonsecca

    2018-01-01

    Schizophrenia is associated with an increased mortality from cardiovascular disease. Relatively few studies have assessed the putative association of schizophrenia pathophysiology with sudden death. Low adenosine levels have been associated with schizophrenia. In cardiology, increased mortality among patients with congestive heart failure has been associated with genetic polymorphisms that potentially lead to lower adenosine levels. Thus, we hypothesize that adenosine could link schizophrenia and cardiovascular mortality, with decreased adenosine levels leading to increased vulnerability to hyperexcitability following hypoxic insults, increasing the odds of fatal arrhythmias. Low adenosine levels might also lead to a small increase in overall mortality rates and a major increase in the sudden death rate. This hypothesis paves the way for further investigation of the increased cardiac mortality associated with schizophrenia. Potentially, a better characterization of adenosine-related mechanisms of sudden death in schizophrenia could lead to new evidence of factors leading to sudden death in the general population. Copyright © 2017. Published by Elsevier Ltd.

  6. Adenylate Energy Charge in Escherichia coli During Growth and Starvation

    PubMed Central

    Chapman, Astrid G.; Fall, Lana; Atkinson, Daniel E.

    1971-01-01

    The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types. PMID:4333317

  7. Staphylococcus aureus synthesizes adenosine to escape host immune responses

    PubMed Central

    Thammavongsa, Vilasack; Kern, Justin W.; Missiakas, Dominique M.

    2009-01-01

    Staphylococcus aureus infects hospitalized or healthy individuals and represents the most frequent cause of bacteremia, treatment of which is complicated by the emergence of methicillin-resistant S. aureus. We examined the ability of S. aureus to escape phagocytic clearance in blood and identified adenosine synthase A (AdsA), a cell wall–anchored enzyme that converts adenosine monophosphate to adenosine, as a critical virulence factor. Staphylococcal synthesis of adenosine in blood, escape from phagocytic clearance, and subsequent formation of organ abscesses were all dependent on adsA and could be rescued by an exogenous supply of adenosine. An AdsA homologue was identified in the anthrax pathogen, and adenosine synthesis also enabled escape of Bacillus anthracis from phagocytic clearance. Collectively, these results suggest that staphylococci and other bacterial pathogens exploit the immunomodulatory attributes of adenosine to escape host immune responses. PMID:19808256

  8. Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression

    PubMed Central

    Headrick, John P; Willems, Laura; Ashton, Kevin J; Holmgren, Kirsten; Peart, Jason; Matherne, G Paul

    2003-01-01

    The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2–4 months) and moderately aged (16–18 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 ± 2 vs. 18 ± 2 mmHg in young) and reduced developed (37 ± 3 vs. 83 ± 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 ± 2 vs. 16 ± 2 IU g−1in young) whereas the incidence of tachyarrhythmias was similar in young (15 ± 1 %) and aged hearts (16 ± 1 %). Functional analysis confirmed equipotent effects of 50 μm adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 μm adenosine improved diastolic (5 ± 1 mmHg) and developed pressures (134 ± 7 mmHg) and LDH loss (6 ± 2 IU g−1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to ∼10 %). In contrast to adenosine, 50 μm diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 μm 5-hydroxydecanoate (5-HD) and 3 μm chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 μm 8-sulfophenyltheophylline) and A1-selective (150 nm 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (∼8 fmol mg−1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring

  9. Adenosine regulation of microtubule dynamics in cardiac hypertrophy.

    PubMed

    Fassett, John T; Xu, Xin; Hu, Xinli; Zhu, Guangshuo; French, Joel; Chen, Yingjie; Bache, Robert J

    2009-08-01

    There is evidence that endogenous extracellular adenosine reduces cardiac hypertrophy and heart failure in mice subjected to chronic pressure overload, but the mechanism by which adenosine exerts these protective effects is unknown. Here, we identified a novel role for adenosine in regulation of the cardiac microtubule cytoskeleton that may contribute to its beneficial effects in the overloaded heart. In neonatal cardiomyocytes, phenylephrine promoted hypertrophy and reorganization of the cytoskeleton, which included accumulation of sarcomeric proteins, microtubules, and desmin. Treatment with adenosine or the stable adenosine analog 2-chloroadenosine, which decreased hypertrophy, specifically reduced accumulation of microtubules. In hypertrophied cardiomyocytes, 2-chloroadenosine or adenosine treatment preferentially targeted stabilized microtubules (containing detyrosinated alpha-tubulin). Consistent with a role for endogenous adenosine in reducing microtubule stability, levels of detyrosinated microtubules were elevated in hearts of CD73 knockout mice (deficient in extracellular adenosine production) compared with wild-type mice (195%, P < 0.05). In response to aortic banding, microtubules increased in hearts of wild-type mice; this increase was exaggerated in CD73 knockout mice, with significantly greater amounts of tubulin partitioning into the cold-stable Triton-insoluble fractions. The levels of this stable cytoskeletal fraction of tubulin correlated strongly with the degree of heart failure. In agreement with a role for microtubule stabilization in promoting cardiac dysfunction, colchicine treatment of aortic-banded mice reduced hypertrophy and improved cardiac function compared with saline-treated controls. These results indicate that microtubules contribute to cardiac dysfunction and identify, for the first time, a role for adenosine in regulating cardiomyocyte microtubule dynamics.

  10. Mechanism-specific effects of adenosine on ventricular tachycardia.

    PubMed

    Lerman, Bruce B; Ip, James E; Shah, Bindi K; Thomas, George; Liu, Christopher F; Ciaccio, Edward J; Wit, Andrew L; Cheung, Jim W; Markowitz, Steven M

    2014-12-01

    There is no universally accepted method by which to diagnose clinical ventricular tachycardia (VT) due to cAMP-mediated triggered activity. Based on cellular and clinical data, adenosine termination of VT is thought to be consistent with a diagnosis of triggered activity. However, a major gap in evidence mitigates the validity of this proposal, namely, defining the specificity of adenosine response in well-delineated reentrant VT circuits. To this end, we systematically studied the effects of adenosine in a model of canine reentrant VT and in human reentrant VT, confirmed by 3-dimensional, pace- and substrate mapping. Adenosine (12 mg [IQR 12-24]) failed to terminate VT in 31 of 31 patients with reentrant VT due to structural heart disease, and had no effect on VT cycle length (age, 67 years [IQR 53-74]); ejection fraction, 35% [IQR 20-55]). In contrast, adenosine terminated VT in 45 of 50 (90%) patients with sustained focal right or left outflow tract tachycardia. The sensitivity of adenosine for identifying VT due to triggered activity was 90% (95% CI, 0.78-0.97) and its specificity was 100% (95% CI, 0.89-1.0). Additionally, reentrant circuits were mapped in the epicardial border zone of 4-day-old infarcts in mongrel dogs. Adenosine (300-400 μg/kg) did not terminate sustained VT or have any effect on VT cycle length. These data support the concept that adenosine's effects on ventricular myocardium are mechanism specific, such that termination of VT in response to adenosine is diagnostic of cAMP-mediated triggered activity. © 2014 Wiley Periodicals, Inc.

  11. Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

    DOEpatents

    Bitensky, Mark W.; Yoshida, Tatsuro

    1997-01-01

    Method using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4.degree. C. storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4.degree. C. for prolonged periods of time is achieved by removing oxygen therefrom at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate.

  12. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

    PubMed Central

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    2016-01-01

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

  13. Role of adenosine receptors in caffeine tolerance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 ofmore » 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.« less

  14. A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

    PubMed

    Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

    2012-03-01

    A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

  15. Inactivation of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii by 2 prime -chloro-2 prime -deoxyuridine 5 prime -triphosphate: A 3 prime -2 prime hydrogen transfer during the formation of 3 prime -keto-2 prime -deoxyuridine 5 prime -triphosphate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ashley, G.W.; Harris, G.; Stubbe, J.

    1988-10-04

    The ribonucleoside triphosphate reductase of Lactobacillus leichmannii converts the substrate analogue 2{prime}-chloro-2{prime}-deoxyuridine 5{prime}-triphosphate (C1UTP) into a mixture of 2{prime}-deoxyuridine triphosphate (dUTP) and the unstable product 3{prime}-keto-2{prime}-deoxyuridine triphosphate (3{prime}-keto-dUTP). This ketone can be trapped by reduction with NaBH{sub 4}, producing a 4:1 mixture of xylo-dUTP and dUTP. When (3{prime}-{sup 3}H)C1UTP is treated with enzyme in the presence of NaBH{sub 4}, the isomeric deoxyuridines isolated after alkaline phosphatase treatment retained 15% of the {sup 3}H in C1UTP. Degradation of these isomeric nucleosides has established the location of the {sup 3}H in 3{prime}-keto-dUTP as predominantly 2{prime}(S). The xylo-dU had 98.6% of its labelmore » at the 2{prime}(S) position and 1.5% at 2{prime}(R). The isolated dU had 89.6% of its label at 2{prime}(S) and 1.4% at 2{prime}(R), with the remaining 9% label inferred to be at the 3{prime}-carbon, this resulting from the direct enzymic production of dUTP. These results are consistent with enzymic production of a 1:1,000 mixture of dUTP and 3{prime}-keto-dUTP, where the 3{prime}-hydrogen of C1UTP is retained at 3{prime} during production of dUTP and is transferred to 2{prime}(S) during production of 3{prime}-keto-dUTP. The implications of these results and the unique role of the cofactor adenosylcobalamin are discussed in terms of reductase being a model for the B{sub 12}-dependent rearrangement reactions.« less

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

    PubMed

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

    2014-03-25

    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.

  17. An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep.

    PubMed

    Bjorness, Theresa E; Dale, Nicholas; Mettlach, Gabriel; Sonneborn, Alex; Sahin, Bogachan; Fienberg, Allen A; Yanagisawa, Masashi; Bibb, James A; Greene, Robert W

    2016-03-30

    Sleep homeostasis reflects a centrally mediated drive for sleep, which increases during waking and resolves during subsequent sleep. Here we demonstrate that mice deficient for glial adenosine kinase (AdK), the primary metabolizing enzyme for adenosine (Ado), exhibit enhanced expression of this homeostatic drive by three independent measures: (1) increased rebound of slow-wave activity; (2) increased consolidation of slow-wave sleep; and (3) increased time constant of slow-wave activity decay during an average slow-wave sleep episode, proposed and validated here as a new index for homeostatic sleep drive. Conversely, mice deficient for the neuronal adenosine A1 receptor exhibit significantly decreased sleep drive as judged by these same indices. Neuronal knock-out of AdK did not influence homeostatic sleep need. Together, these findings implicate a glial-neuronal circuit mediated by intercellular Ado, controlling expression of homeostatic sleep drive. Because AdK is tightly regulated by glial metabolic state, our findings suggest a functional link between cellular metabolism and sleep homeostasis. The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets more directly related to

  18. Increased cortical extracellular adenosine correlates with seizure termination.

    PubMed

    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

    2014-02-01

    Seizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent on neurotransmitters of which little is known regarding their periictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Furthermore, microdialysis studies in humans suggest that adenosine is elevated periictally, 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. 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 voltammetry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine-specific triangular waveform or biosensors, respectively. Simultaneous ECoG and electrochemistry demonstrated an average adenosine increase of 260% compared to baseline, at 7.5 ± 16.9 s with amperometry (n = 75 events) and 2.6 ± 11.2 s 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. 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. Wiley Periodicals, Inc. © 2014 International League Against

  19. CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca(2+)-ATPase.

    PubMed

    Komuro, Yasuaki; Re, Suyong; Kobayashi, Chigusa; Muneyuki, Eiro; Sugita, Yuji

    2014-09-09

    Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure-function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca(2+)-ATPase and four soluble proteins. The ATP-bound structure of Ca(2+)-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins.

  20. A Role for Adenosine Deaminase in Drosophila Larval Development

    PubMed Central

    Dolezal, Tomas; Dolezelova, Eva; Zurovec, Michal

    2005-01-01

    Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals. PMID:15907156

  1. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release

    PubMed Central

    Nguyen, Michael D.; Venton, B. Jill

    2014-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future. PMID:26900429

  2. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release.

    PubMed

    Nguyen, Michael D; Venton, B Jill

    2015-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future.

  3. 3-phosphoglycerate kinase from Hydrogenomonas facilis.

    NASA Technical Reports Server (NTRS)

    Mcfadden, B. A.; Schuster, E.

    1972-01-01

    Description of studies of the kinetics of heat inactivation of phosphoglycerate kinase in the soluble fraction from Hydrogenomonas facilis, its extensive purification, and inhibition by adenosine monophosphate (AMP). No evidence was found for an enzyme which catalyzes adenosine-triphosphate-dependent conversion of 3-phosphoglycerate to 1,3-diphosphoglycerate, AMP, and phosphate.

  4. Adenosine receptors and caffeine in retinopathy of prematurity.

    PubMed

    Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

    2017-06-01

    Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A 1 R, A 2A R, A 2B R) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Adenosine receptors and caffeine in retinopathy of prematurity

    PubMed Central

    Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

    2017-01-01

    Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A1R, A2AR, A2BR) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. PMID:28088487

  6. Application of the newly developed Japanese adenosine normal database for adenosine stress myocardial scintigraphy.

    PubMed

    Harata, Shingo; Isobe, Satoshi; Morishima, Itsuro; Suzuki, Susumu; Tsuboi, Hideyuki; Sone, Takahito; Ishii, Hideki; Murohara, Toyoaki

    2015-10-01

    The currently available Japanese normal database (NDB) in stress myocardial perfusion scintigraphy recommended by the Japanese Society of Nuclear Medicine (JSNM-NDB) is created based on the data from exercise tests. The newly developed adenosine normal database (ADS-NDB) remains to be validated for patients undergoing adenosine stress test. We tested whether the diagnostic accuracy of adenosine stress test is improved by the use of ADS-NDB (Kanazawa University). Of 233 consecutive patients undergoing (99m)Tc-MIBI adenosine stress test, 112 patients were tested. The stress/rest myocardial (99m)Tc-MIBI single-photon emission computed tomography (SPECT) images were analyzed by AutoQUANT 7.2 with both ADS-NDB and JSNM-NDB. The summed stress score (SSS) and summed difference score (SDS) were calculated. The agreements of the post-stress defect severity between ADS-NDB and JSNM-NDB were assessed using a weighted kappa statistic. In all patients, mean SSSs of all, right coronary artery (RCA), left anterior descending (LAD), and left circumflex (LCx) territories were significantly lower with ADS-NDB than those with JSNM-NDB. Mean SDSs in all, RCA, and LAD territories were significantly lower with ADS-NDB than those with JSNM-NDB. In 28 patients with significant coronary stenosis, the mean SSS in the RCA territory was significantly lower with ADS-NDB than that with JSNM-NDB. In 84 patients without ischemia, both mean SSSs and SDSs in all, RCA, LAD, and LCx territories were significantly lower with ADS-NDB than those with JSNM-NDB. Weighted kappa values of all patients, patients with significant stenosis, and patients without ischemia were 0.89, 0.83, and 0.92, respectively. Differences were observed between results from ADS-NDB and JSNM-NDB. The diagnostic accuracy of adenosine stress myocardial perfusion scintigraphy may be improved by reducing false-positive results.

  7. Adenosine signaling contributes to ethanol-induced fatty liver in mice

    PubMed Central

    Peng, Zhongsheng; Borea, Pier Andrea; Wilder, Tuere; Yee, Herman; Chiriboga, Luis; Blackburn, Michael R.; Azzena, Gianfranco; Resta, Giuseppe; Cronstein, Bruce N.

    2009-01-01

    Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical 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:19221436

  8. Adenosine signaling in normal and sickle erythrocytes and beyond.

    PubMed

    Zhang, Yujin; Xia, Yang

    2012-08-01

    Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications. In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A(2B) receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O(2) release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A(2A) receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD. Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and

  9. Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

    PubMed

    Akinyemi, Ayodele Jacob; Thomé, Gustavo Roberto; Morsch, Vera Maria; Bottari, Nathieli B; Baldissarelli, Jucimara; de Oliveira, Lizielle Souza; Goularte, Jeferson Ferraz; Belló-Klein, Adriane; Oboh, Ganiyu; Schetinger, Maria Rosa Chitolina

    2016-07-01

    Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of l-NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l-NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension-derived complications associated to platelet hyperactivity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Method for extending the useful shelf-life of refrigerated red blood cells by flushing with inert gas

    DOEpatents

    Bitensky, M.W.; Yoshida, Tatsuro

    1997-04-29

    A method is disclosed using oxygen removal for extending the useful shelf-life of refrigerated red blood cells. A cost-effective, 4 C storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. Preservation of adenosine triphosphate levels and reduction in hemolysis and in membrane vesicle production of red blood cells stored at 4 C for prolonged periods of time is achieved by removing oxygen from the red blood cells at the time of storage; in particular, by flushing with an inert gas. Adenosine triphosphate levels of the stored red blood cells are boosted in some samples by addition of ammonium phosphate. 4 figs.

  11. 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

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

    USGS Publications Warehouse

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

    2009-01-01

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

  13. Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani.

    PubMed

    Bhaumik, D; Datta, A K

    1988-04-01

    The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.

  14. Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.

    PubMed

    Schindler, Charles W; Karcz-Kubicha, Marzena; Thorndike, Eric B; Müller, Christa E; Tella, Srihari R; Ferré, Sergi; Goldberg, Steven R

    2005-03-01

    1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing

  15. Dual activity of certain HIT-proteins: A. thaliana Hint4 and C. elegans DcpS act on adenosine 5'-phosphosulfate as hydrolases (forming AMP) and as phosphorylases (forming ADP).

    PubMed

    Guranowski, Andrzej; Wojdyła, Anna Maria; Zimny, Jarosław; Wypijewska, Anna; Kowalska, Joanna; Jemielity, Jacek; Davis, Richard E; Bieganowski, Paweł

    2010-01-04

    Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5'-phosphosulfate (APS or SO(4)-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.

  16. Regulation of the calcium release channel from rabbit skeletal muscle by the nucleotides ATP, AMP, IMP and adenosine

    PubMed Central

    Laver, Derek R; Lenz, Gerlinde K E; Lamb, Graham D

    2001-01-01

    Nucleotide activation of skeletal muscle ryanodine receptors (RyRs) was studied in planar lipid bilayers in order to understand RyR regulation in vivo under normal and fatigued conditions. With ‘resting’ calcium (100 nm cytoplasmic and 1 mm luminal), RyRs had an open probability (Po) of ∼0.01 in the absence of nucleotides and magnesium. ATP reversibly activated RyRs with Po at saturation (Pmax) ∼0.33 and Ka (concentration for half-maximal activation) ∼0.36 mm and with a Hill coefficient (nH) of ∼1.8 in RyRs when Pmax < 0.5 and ∼4 when Pmax > 0.5. AMP was a much weaker agonist (Pmax∼0.09) and adenosine was weaker still (Pmax∼0.01–0.02), whereas inosine monophosphate (IMP), the normal metabolic end product of ATP hydrolysis, produced no activation at all. Adenosine acted as a competitive antagonist that reversibly inhibited ATP- and AMP-activated RyRs with nH∼1 and Ki∼0.06 mm at [ATP] < 0.5 mm, increasing 4-fold for each 2-fold increase in [ATP] above 0.5 mm. This is explained by the binding of a single adenosine preventing the cooperative binding of two ATP or AMP molecules, with dissociation constants of 0.4, 0.45 and 0.06 mm for ATP, AMP and adenosine, respectively. Importantly, IMP (≤ 8 mm) had no inhibitory effect whatsoever on ATP-activated RyRs. Mean open (τo) and closed (τc) dwell-times were more closely related to Po than to the nucleotide species or individual RyRs. At Po < 0.2, RyR regulation occurred via changes in τc, whereas at higher Po this also occurred via changes in τo. The detailed properties of activation and competitive inhibition indicated complex channel behaviour that could be explained in terms of a model involving interactions between different subunits of the RyR homotetramer. The results also show how deleterious adenosine accumulation is to the function of RyRs in skeletal muscle and, by comparison with voltage sensor-controlled Ca2+ release, indicate that voltage sensor activation requires ATP binding to

  17. Formation of adenosine 5'-tetraphosphate from the acyl phosphate intermediate: a difference between the MurC and MurD synthetases of Escherichia coli.

    PubMed

    Bouhss, A; Dementin, S; van Heijenoort, J; Parquet, C; Blanot, D

    1999-06-18

    The mechanism of the Mur synthetases of peptidoglycan biosynthesis is thought to involve in each case the successive formation of an acyl phosphate and a tetrahedral intermediate. The existence of the acyl phosphates for the MurC and MurD enzymes from Escherichia coli was firmly established by their in situ reduction by sodium borohydride followed by acid hydrolysis, yielding the corresponding amino alcohols. Furthermore, it was found that MurD, but not MurC, catalyses the synthesis of adenosine 5'-tetraphosphate from the acyl phosphate, thereby substantiating its existence and pointing out a difference between the two enzymes.

  18. Correlation of transient adenosine release and oxygen changes in the caudate-putamen

    PubMed Central

    Wang, Ying; Venton, B. Jill

    2016-01-01

    Adenosine is an endogenous nucleoside that modulates important physiological processes, such as vasodilation, in the central nervous system. A rapid, 2–4 seconds, mode of adenosine signaling has been recently discovered, but the relationship between this type of adenosine and blood flow change has not been characterized. In this study, adenosine and oxygen changes were simultaneously measured using fast-scan cyclic voltammetry. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. About 34% of adenosine transients in the rat caudate-putamen are correlated with a subsequent transient change in oxygen. The amount of oxygen was correlated with the concentration of adenosine release and larger adenosine transients (over 0.4 μM) always had subsequent oxygen changes. The average duration of adenosine and oxygen transients were 3.2 seconds and 3.5 seconds, respectively. On average, the adenosine release starts and peaks 0.2 seconds prior to the oxygen. The A2a antagonist, SCH442416, decreased the number of both adenosine and oxygen transient events by about 32%. However, the A1 antagonist, DPCPX, did not significantly affect simultaneous adenosine and oxygen release. The nitric oxide (NO) synthase inhibitor L-NAME also did not affect the concentration or number of adenosine and oxygen release events. These results demonstrate that both adenosine and oxygen release are modulated via A2a receptors. The correlation of transient concentrations, time delay between adenosine and oxygen peaks, and effect of A2a receptors suggests adenosine modulates blood flow on a rapid, sub-second time scale. PMID:27314215

  19. Ticagrelor and Rosuvastatin Have Additive Cardioprotective Effects via Adenosine.

    PubMed

    Birnbaum, Yochai; Birnbaum, Gilad D; Birnbaum, Itamar; Nylander, Sven; Ye, Yumei

    2016-12-01

    Ticagrelor inhibits the equilibrative-nucleoside-transporter-1 and thereby, adenosine cell re-uptake. Ticagrelor limits infarct size (IS) in non-diabetic rats and the effect is adenosine-dependent. Statins, via ecto-5'-nucleotidase activation, also increase adenosine levels and limit IS. Ticagrelor and rosuvastatin have additive effects on myocardial adenosine levels, and therefore, on IS and post-reperfusion activation of the NLRP3-inflammasome. Diabetic ZDF rats received via oral gavage; water (control), ticagrelor (150 mg/kg/d), prasugrel (7.5 mg/kg/d), rosuvastatin (5 mg/kg/d), ticagrelor + rosuvastatin and prasugrel + rosuvastatin for 3d. On day 4, rats underwent 30 min coronary artery occlusion and 24 h of reperfusion. Two additional groups received, ticagrelor + rosuvastatin or water in combination with CGS15943 (CGS, an adenosine receptor antagonist, 10 mg/kg i.p. 1 h before ischemia). Both ticagrelor and rosuvastatin increased myocardial adenosine levels with an additive effect of the combination whereas prasugrel had no effect. Similarly, both ticagrelor and rosuvastatin significantly reduced IS with an additive effect of the combination whereas prasugrel had no effect. The effect on IS was adenosine dependent as CGS15943 reversed the effect of ticagrelor + rosuvastatin. The ischemia-reperfusion injury increased myocardial mRNA levels of NLRP3, ASC, IL-1β and IL-6. Ticagrelor and rosuvastatin, but not prasugrel, significantly decreased these pro-inflammatory mediators with a trend to an additive effect of the combination. The combination also increased the levels of anti-inflammatory 15-epilipoxin A 4 . Ticagrelor and rosuvastatin when given in combination have an additive effect on local myocardial adenosine levels in the setting of ischemia reperfusion. This translates into an additive cardioprotective effect mediated by adenosine-induced effects including downregulation of pro- but upregulation of anti-inflammatory mediators.

  20. 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

  1. Detrimental effects of adenosine signaling in sickle cell disease.

    PubMed

    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

    2011-01-01

    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 A(2B) receptor (A(2B)R)-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 A(2B)R has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.

  2. The alterations in adenosine nucleotides and lactic acid in striated muscles of rats during Rigor mortis following death with drowning or cervical dislocation.

    PubMed

    Pençe, Halime Hanim; Pençe, Sadrettin; Kurtul, Naciye; Yilmaz, Necat; Kocoglu, Hasan; Bakan, Ebubekir

    2003-01-01

    In this study, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and lactic acid in the muscles of masseter, triceps, and quadriceps obtained from right and left sides of Spraque-Dawley rats following death were investigated. The samples were taken immediately and 120 minutes after death occurred. The rats were killed either by cervical dislocation or drowning. ATP concentrations in the muscles of masseter, triceps, and quadriceps were lower in samples obtained 120 minutes after death than in those obtained immediately after death. ADP, AMP, and lactic acid concentrations in these muscles were higher in samples obtained 120 minutes after death than those obtained immediately after death. A positive linear correlation was determined between ATP and ADP concentrations in quadriceps muscles of the rats killed with cervical dislocation and in triceps muscles of the rats killed with drowning. When rats killed with cervical dislocation and with drowning were compared, ADP, AMP, and lactic acid concentrations were lower in the former than in the latter for both times (immediately and 120 minutes after death occurred). In the case of drowning, ATP is consumed faster because of hard exercise or severe physical activity, resulting in a faster rigor mortis. Higher lactic acid levels were determined in muscles of the rats killed with drowning than the other group. In the control and electric shock rats, ATP decreased in different levels in the three different muscle types mentioned above in control group, being much decline in masseter and then in quadriceps. This may be caused by lower mass and less glycogen storage of masseter. No different ATP levels were measured in drowning group with respect to the muscle type possibly because of the severe activity of triceps and quadriceps and because of smaller mass of masseter. One can conclude that the occurrence of rigor mortis is closely related to the mode of death.

  3. Elevated placental adenosine signaling contributes to the pathogenesis of preeclampsia.

    PubMed

    Iriyama, Takayuki; Sun, Kaiqi; Parchim, Nicholas F; Li, Jessica; Zhao, Cheng; Song, Anren; Hart, Laura A; Blackwell, Sean C; Sibai, Baha M; Chan, Lee-Nien L; Chan, Teh-Sheng; Hicks, M John; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2015-02-24

    Preeclampsia is a prevalent hypertensive disorder of pregnancy and a leading cause of maternal and neonatal morbidity and mortality worldwide. This pathogenic condition is speculated to be caused by placental abnormalities that contribute to the maternal syndrome. However, the specific factors and signaling pathways that lead to impaired placentas and maternal disease development remain elusive. Using 2 independent animal models of preeclampsia (genetically engineered pregnant mice with elevated adenosine exclusively in placentas and a pathogenic autoantibody-induced preeclampsia mouse model), we demonstrated that chronically elevated placental adenosine was sufficient to induce hallmark features of preeclampsia, including hypertension, proteinuria, small fetuses, and impaired placental vasculature. Genetic and pharmacological approaches revealed that elevated placental adenosine coupled with excessive A₂B adenosine receptor (ADORA2B) signaling contributed to the development of these features of preeclampsia. Mechanistically, we provided both human and mouse evidence that elevated placental CD73 is a key enzyme causing increased placental adenosine, thereby contributing to preeclampsia. We determined that elevated placental adenosine signaling is a previously unrecognized pathogenic factor for preeclampsia. Moreover, our findings revealed the molecular basis underlying the elevation of placental adenosine and the detrimental role of excess placental adenosine in the pathophysiology of preeclampsia, and thereby, we highlight novel therapeutic targets. © 2014 American Heart Association, Inc.

  4. Mechanism of conformational coupling in SecA: Key role of hydrogen-bonding networks and water interactions.

    PubMed

    Milenkovic, Stefan; Bondar, Ana-Nicoleta

    2016-02-01

    SecA uses the energy yielded by the binding and hydrolysis of adenosine triphosphate (ATP) to push secretory pre-proteins across the plasma membrane in bacteria. Hydrolysis of ATP occurs at the nucleotide-binding site, which contains the conserved carboxylate groups of the DEAD-box helicases. Although crystal structures provide valuable snapshots of SecA along its reaction cycle, the mechanism that ensures conformational coupling between the nucleotide-binding site and the other domains of SecA remains unclear. The observation that SecA contains numerous hydrogen-bonding groups raises important questions about the role of hydrogen-bonding networks and hydrogen-bond dynamics in long-distance conformational couplings. To address these questions, we explored the molecular dynamics of SecA from three different organisms, with and without bound nucleotide, in water. By computing two-dimensional hydrogen-bonding maps we identify networks of hydrogen bonds that connect the nucleotide-binding site to remote regions of the protein, and sites in the protein that respond to specific perturbations. We find that the nucleotide-binding site of ADP-bound SecA has a preferred geometry whereby the first two carboxylates of the DEAD motif bridge via hydrogen-bonding water. Simulations of a mutant with perturbed ATP hydrolysis highlight the water-bridged geometry as a key structural element of the reaction path. Copyright © 2015. Published by Elsevier B.V.

  5. Adenosine uptake by the isolated epithelium of guine pig jejunum.

    PubMed

    Kolassa, N; Stengg, R; Turnheim, K

    1977-10-01

    The uptake of [8-14C]adenosine by the isolated epithelium of guinea pig jejunum was faster than that of inosine, hypoxanthine, or adenine. The initial velocity of adenosine uptake from both the luminal and the antiluminal side of the epithelium exhibited saturation kinetics. The apparent Km, V, and passive permeability of luminal adenosine uptake were all lower than the corresponding values of antiluminal uptake. p-Nitrobenzyl-thioguanosine inhibited adenosine uptake from both the luminal and the antiluminal side, whilst hexobendine decreased the uptake only from the antiluminal side of the epithelium. The results suggest that adenosine enters the intestinal epithelium by a carrier-mediated process in addition to passive diffusion. The antiluminal transport system for adenosine seems similar to that of other tissues with respect to hexobendine inhibition; the luminal transport mechanism, however, exhibits different properties, being insensitive to hexobendine.

  6. Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).

    PubMed

    Magalhães, Luana; de Oliveira, Arthur Henrique Cavalcante; de Souza Vasconcellos, Raphael; Mariotini-Moura, Christiane; de Cássia Firmino, Rafaela; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lúcia

    2016-01-01

    Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8mmolL(-1) for the ADP and ATP substrates, respectively. Suramin (1mmolL(-1)) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.

    PubMed

    McDermott, Catherine; Chess-Williams, Russ; Grant, Gary D; Perkins, Anthony V; McFarland, Amelia J; Davey, Andrew K; Anoopkumar-Dukie, Shailendra

    2012-03-01

    We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier

  8. Reentry Tachycardia in Children: Adenosine Can Make It Worse.

    PubMed

    Hien, Maximilian D; Benito Castro, Fernando; Fournier, Philippe; Filleron, Anne; Tran, Tu-Anh

    2016-10-08

    We report on a rare but severe complication of adenosine use in a child with reentry tachycardia. Treatment with adenosine, which is the standard medical therapy of atrioventricular reentry tachycardia, led to the development of an irregular wide complex tachycardia, caused by rapid ventricular response to atrial fibrillation. The girl was finally stabilized with electrical cardioversion. We analyze the pathomechanism and discuss possible treatment options. Atrial fibrillation, as well as its conduction to the ventricles, can be caused by adenosine. Rapid ventricular response in children with Wolff-Parkinson-White syndrome is more frequent than previously believed. A patient history of atrial fibrillation is a contraindication for cardioversion with adenosine and needs to be assessed in children with reentry tachycardia. High-risk patients may potentially profit from prophylactic comedication with antiarrhythmic agents, such as flecainide, ibutilide, or vernakalant, before adenosine administration.

  9. TOR-induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease.

    PubMed

    Detke, Siegfried

    2007-05-15

    TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain, which was recognized by TOR in this internalization pathway, was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature-sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking.

  10. Beneficial and detrimental role of adenosine signaling in diseases and therapy

    PubMed Central

    Liu, Hong

    2015-01-01

    Adenosine is a major signaling nucleoside that orchestrates cellular and tissue adaptation under energy depletion and ischemic/hypoxic conditions by activation of four G protein-coupled receptors (GPCR). The regulation and generation of extracellular adenosine in response to stress are critical in tissue protection. Both mouse and human studies reported that extracellular adenosine signaling plays a beneficial role during acute states. However, prolonged excess extracellular adenosine is detrimental and contributes to the development and progression of various chronic diseases. In recent years, substantial progress has been made to understand the role of adenosine signaling in different conditions and to clarify its significance during the course of disease progression in various organs. These efforts have and will identify potential therapeutic possibilities for protection of tissue injury at acute stage by upregulation of adenosine signaling or attenuation of chronic disease progression by downregulation of adenosine signaling. This review is to summarize current progress and the importance of adenosine signaling in different disease stages and its potential therapeutic effects. PMID:26316513

  11. Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

    PubMed

    Kiechle, F L; Sykes, E; Artiss, J D

    1995-01-01

    Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Awwad, Khaldeyah; Desai, Anna; Smith, Clyde

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

  13. Role of inositol 1,4,5-triphosphate signalling in gravitropic and phototropic gene expression.

    PubMed

    Salinas-Mondragon, Raul E; Kajla, Jyoti D; Perera, Imara Y; Brown, Christopher S; Sederoff, Heike Winter

    2010-12-01

    Plants sense light and gravity to orient their direction of growth. One common component in the early events of both phototropic and gravitropic signal transduction is activation of phospholipase C (PLC), which leads to an increase in inositol 1,4,5-triphosphate (InsP(3)) levels. The InsP(3) signal is terminated by hydrolysis of InsP(3) through inositolpolyphosphate-5-phosphatases (InsP 5-ptases). Arabidopsis plants expressing a heterologous InsP 5-ptase have low basal InsP(3) levels and exhibit reduced gravitropic and phototropic bending. Downstream effects of InsP(3)-mediated signalling are not understood. We used comparative transcript profiling to characterize gene expression changes in gravity- or light-stimulated Arabidopsis root apices that were manipulated in their InsP(3) metabolism either through inhibition of PLC activity or expression of InsP 5-ptase. We identified InsP(3)-dependent and InsP(3)-independent co-regulated gene sets in response to gravity or light stimulation. Inhibition of PLC activity in wild-type plants caused similar changes in transcript abundance in response to gravitropic and phototropic stimulation as in the transgenic lines. Therefore, we conclude that changes in gene expression in response to gravitropic and phototropic stimulation are mediated by two signal transduction pathways that vary in their dependence on changes in InsP(3). © 2010 Blackwell Publishing Ltd.

  14. Low-dose adenosine stress echocardiography: detection of myocardial viability.

    PubMed

    Djordjevic-Dikic, Ana; Ostojic, Miodrag; Beleslin, Branko; Nedeljkovic, Ivana; Stepanovic, Jelena; Stojkovic, Sinisa; Petrasinovic, Zorica; Nedeljkovic, Milan; Saponjski, Jovica; Giga, Vojislav

    2003-06-03

    The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals) echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of >or= 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 +/- 2 months) were available in 24 revascularized patients. Wall motion score index improved from rest 1.55 +/- 0.30 to 1.33 +/- 0.26 at low-dose adenosine (p < 0.001). Of the 257 segments with baseline dyssynergy, adenosine echocardiography identified 122 segments as positive for viability, and 135 as necrotic since no improvement of systolic thickening was observed. Follow-up wall motion score index was 1.31 +/- 0.30 (p < 0.001 vs. rest). The sensitivity of adenosine echo test for identification of viable segments was 87%, while specificity was 95%, and diagnostic accuracy 90%. Positive and negative predictive values were 97% and 80%, respectively. Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability.

  15. Low-dose adenosine stress echocardiography: Detection of myocardial viability

    PubMed Central

    Djordjevic-Dikic, Ana; Ostojic, Miodrag; Beleslin, Branko; Nedeljkovic, Ivana; Stepanovic, Jelena; Stojkovic, Sinisa; Petrasinovic, Zorica; Nedeljkovic, Milan; Saponjski, Jovica; Giga, Vojislav

    2003-01-01

    Objective The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Background Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Methods Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals) echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of ≥ 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 ± 2 months) were available in 24 revascularized patients. Results Wall motion score index improved from rest 1.55 ± 0.30 to 1.33 ± 0.26 at low-dose adenosine (p < 0.001). Of the 257 segments with baseline dyssynergy, adenosine echocardiography identified 122 segments as positive for viability, and 135 as necrotic since no improvement of systolic thickening was observed. Follow-up wall motion score index was 1.31 ± 0.30 (p < 0.001 vs. rest). The sensitivity of adenosine echo test for identification of viable segments was 87%, while specificity was 95%, and diagnostic accuracy 90%. Positive and negative predictive values were 97% and 80%, respectively. Conclusion Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability. PMID:12812523

  16. 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.

  17. Adenosine signaling promotes regeneration of pancreatic β-cells in vivo

    PubMed Central

    Andersson, Olov; Adams, Bruce A.; Yoo, Daniel; Ellis, Gregory C.; Gut, Philipp; Anderson, Ryan M.; German, Michael S.; Stainier, Didier Y. R.

    2012-01-01

    Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes. PMID:22608007

  18. A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

    PubMed Central

    Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

    2015-01-01

    Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

  19. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  20. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

  1. Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

    PubMed Central

    Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

    2012-01-01

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

  2. Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

    PubMed

    Arulanandam, Bernard P; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J; Chambers, James P

    2012-10-26

    Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

  3. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors.

    PubMed

    Sun, D; Samuelson, L C; Yang, T; Huang, Y; Paliege, A; Saunders, T; Briggs, J; Schnermann, J

    2001-08-14

    Adenosine is a determinant of metabolic control of organ function increasing oxygen supply through the A2 class of adenosine receptors and reducing oxygen demand through A1 adenosine receptors (A1AR). In the kidney, activation of A1AR in afferent glomerular arterioles has been suggested to contribute to tubuloglomerular feedback (TGF), the vasoconstriction elicited by elevations in [NaCl] in the macula densa region of the nephron. To further elucidate the role of A1AR in TGF, we have generated mice in which the entire A1AR coding sequence was deleted by homologous recombination. Homozygous A1AR mutants that do not express A1AR mRNA transcripts and do not respond to A1AR agonists are viable and without gross anatomical abnormalities. Plasma and urinary electrolytes were not different between genotypes. Likewise, arterial blood pressure, heart rates, and glomerular filtration rates were indistinguishable between A1AR(+/+), A1AR(+/-), and A1AR(-/-) mice. TGF responses to an increase in loop of Henle flow rate from 0 to 30 nl/min, whether determined as change of stop flow pressure or early proximal flow rate, were completely abolished in A1AR(-/-) mice (stop flow pressure response, -6.8 +/- 0.55 mmHg and -0.4 +/- 0.2 in A1AR(+/+) and A1AR(-/-) mice; early proximal flow rate response, -3.4 +/- 0.4 nl/min and +0.02 +/- 0.3 nl/min in A1AR(+/+) and A1AR(-/-) mice). Absence of TGF responses in A1AR-deficient mice suggests that adenosine is a required constituent of the juxtaglomerular signaling pathway. A1AR null mutant mice are a promising tool to study the functional role of A1AR in different target tissues.

  4. 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

  5. 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. Copyright © 2016 The Author(s).

  6. 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.

  7. Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue.

    PubMed

    Strouch, Marci B; Jackson, Edwin K; Mi, Zaichuan; Metes, Nicole A; Carey, Gale B

    2005-06-01

    Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 microL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 microM isoproterenol, or 10 microM isoproterenol plus 1 mM alpha,beta-methylene adenosine 5'-diphosphate (AMPCP), a 5'-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 microM isoproterenol, or 1 microM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.

  8. The hydrolysis of polyimides

    NASA Technical Reports Server (NTRS)

    Hoagland, P. D.; Fox, S. W.

    1973-01-01

    Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.

  9. TOR induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease

    PubMed Central

    Detke, Siegfried

    2007-01-01

    TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain which was recognized by TOR in this internalization pathway was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking. PMID:17428463

  10. Genetics Home Reference: generalized arterial calcification of infancy

    MedlinePlus

    ... helps break down a molecule called adenosine triphosphate (ATP), specifically when it is found outside the cell (extracellular). Extracellular ATP is quickly broken down into other molecules called ...

  11. Adenosine for postoperative analgesia: A systematic review and meta-analysis

    PubMed Central

    2017-01-01

    Purpose Perioperative infusion of adenosine has been suggested to reduce the requirement for inhalation anesthetics, without causing serious adverse effects in humans. We conducted a meta-analysis of randomized controlled trials evaluating the effect of adenosine on postoperative analgesia. Methods We retrieved articles in computerized searches of Scopus, Web of Science, PubMed, EMBASE, and Cochrane Library databases, up to July 2016. We used adenosine, postoperative analgesia, and postoperative pain(s) as key words, with humans, RCT, and CCT as filters. Data of eligible studies were extracted, which included pain scores, cumulative opioid consumption, adverse reactions, and vital signs. Overall incidence rates, relative risk (RR), and 95% confidence intervals (CI) were calculated employing fixed-effects or random-effects models, depending on the heterogeneity of the included trials. Results In total, 757 patients from 9 studies were included. The overall effect of adenosine on postoperative VAS/VRS scores and postoperative opioid consumption was not significantly different from that of controls (P >0.1). The occurrence of PONV and pruritus was not statistically significantly different between an adenosine and nonremifentanil subgroup (P >0.1), but the rate of PONV occurrence was greater in the remifentanil subgroup (P <0.01). Time to first postoperative analgesic requirement in the adenosine group was not significantly difference from that of the saline group (SMD = 0.07, 95%CI: −0.28 to 0.41, P = 0.71); but this occurred significantly later than with remifentanil (SMD = 1.10, 95%CI: 2.48 to 4.06, P < 0.01). Time to hospital discharge was not significantly different between the control and adenosine groups (P = 0.78). The perioperative systolic blood pressure was significantly lower in the adenosine than in the control group in the mannitol subgroup (P < 0.01). The incidence of bradycardia, transient first- degree atrioventricular block, and tachycardia was not

  12. Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

    PubMed

    Li, Ning; Csepe, Thomas A; Hansen, Brian J; Sul, Lidiya V; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Biesiadecki, Brandon J; Hummel, John D; Weiss, Raul; Fedorov, Vadim V

    2016-08-09

    Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. © 2016 American Heart Association, Inc.

  13. ST 1535: a preferential A2A adenosine receptor antagonist.

    PubMed

    Stasi, Maria Antonietta; Borsini, Franco; Varani, Katia; Vincenzi, Fabrizio; Di Cesare, Maria Assunta; Minetti, Patrizia; Ghirardi, Orlando; Carminati, Paolo

    2006-10-01

    Antagonism of the A2A adenosine function has proved beneficial in the treatment of Parkinson's disease, in that it increases L-dopa therapeutical effects without concomitant worsening of its side-effects. In this paper we describe a preferential A2A adenosine antagonist, ST 1535, with long-lasting pharmacodynamic effects. It competitively antagonizes the effects of the A2A adenosine agonist NECA on cAMP in cells cloned with the human A2A adenosine receptor (IC50=353+/-30 nM), and the effects of the A1 adenosine agonist CHA on cAMP in cells cloned with the human A1 adenosine receptor (IC50=510+/-38 nM). ST 1535, at oral doses of 5 and 10 mg/kg, antagonizes catalepsy induced by intracerebroventricular administration of the A2A adenosine agonist CGS 21680 (10 microg/5 microl) in mice. At oral doses ranging between 5 and 20 mg/kg, ST 1535 induces hypermotility and antagonizes haloperidol-induced catalepsy in mice up to 7 h. Oral ST 1535, at 1.25 and 2.5 mg/kg, potentiates L-dopa effects in reducing haloperidol-induced catalepsy. ST 1535 represents a potential new compound, with long-lasting activity, for the treatment of Parkinson's disease.

  14. Mathematical modeling of chemotaxis and glial scarring around implanted electrodes

    NASA Astrophysics Data System (ADS)

    Silchenko, Alexander N.; Tass, Peter A.

    2015-02-01

    It is well known that the implantation of electrodes for deep brain stimulation or microelectrode probes for the recording of neuronal activity is always accompanied by the response of the brain’s immune system leading to the formation of a glial scar around the implantation sites. The implantation of electrodes causes massive release of adenosine-5‧-triphosphate (ATP) and different cytokines into the extracellular space and activates the microglia. The released ATP and the products of its hydrolysis, such as ADP and adenosine, become the main elements mediating chemotactic sensitivity and motility of microglial cells via subsequent activation of P2Y2,12 as well as A3A/A2A adenosine receptors. The size and density of an insulating sheath around the electrode, formed by microglial cells, are important criteria for the optimization of the signal-to-noise ratio during microelectrode recordings or parameters of electrical current delivered to the brain tissue. Here, we study a purinergic signaling pathway underlying the chemotactic motion of microglia towards implanted electrodes as well as the possible impact of an anti-inflammatory coating consisting of the interleukin-1 receptor antagonist. We present a model describing the formation of a stable aggregate around the electrode due to the joint chemo-attractive action of ATP and ADP and the mixed influence of extracellular adenosine. The bioactive coating is modeled as a source of chemo-repellent located near the electrode surface. The obtained analytical and numerical results allowed us to reveal the dependences of size and spatial location of the insulating sheath on the amount of released ATP and estimate the impact of immune suppressive coating on the scarring process.

  15. TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses

    PubMed Central

    Cohen, Heather B.; Briggs, Katharine T.; Marino, John P.; Ravid, Katya; Robson, Simon C.

    2013-01-01

    Sepsis is a highly fatal disease caused by an initial hyperinflammatory response followed by a state of profound immunosuppression. Although it is well appreciated that the initial production of proinflammatory cytokines by macrophages accompanies the onset of sepsis, it remains unclear what causes the transition to an immunosuppressive state. In this study, we reveal that macrophages themselves are key regulators of this transition and that the surface enzyme CD39 plays a critical role in self-limiting the activation process. We demonstrate that Toll-like receptor (TLR)-stimulated macrophages modulate their activation state by increasing the synthesis and secretion of adenosine triphosphate (ATP). This endogenous ATP is paradoxically immunosuppressive due to its rapid catabolism into adenosine by CD39. Macrophages lacking CD39 are unable to transition to a regulatory state and consequently continue to produce inflammatory cytokines. The importance of this transition is demonstrated in a mouse model of sepsis, where small numbers of CD39-deficient macrophages were sufficient to induce lethal endotoxic shock. Thus, these data implicate CD39 as a key “molecular switch” that allows macrophages to self-limit their activation state. We propose that therapeutics targeting the release and hydrolysis of ATP by macrophages may represent new ways to treat inflammatory diseases. PMID:23908469

  16. Nonenzymatic glycation of guanosine 5'-triphosphate by glyceraldehyde: an in vitro study of AGE formation.

    PubMed

    Li, Yuyuan; Dutta, Udayan; Cohenford, Menashi A; Dain, Joel A

    2007-12-01

    Guanosine 5'-triphosphate (GTP) plays a significant role in the bioenergetics, metabolism, and signaling of cells; consequently, any modifications to the structure of the molecule can have profound effects on a cell's survival and function. Previous studies in our laboratory demonstrated that like proteins, purines, and pyrimidines can nonenzymatically react with sugars to generate advanced glycation endproducts (AGEs) and that these AGEs can form in vitro under physiological conditions. The objective of this investigation was twofold. First, it was to evaluate the susceptibility of ATP, GTP, CTP, and TTP to nonenzymatic modification by D-glucose and DL-glyceraldehyde, and second to assess the effect of various factors such as temperature, pH and incubation time, and sugar concentration on the rate and extent of nucleotide triphosphate AGE formation. Of the four nucleotide triphosphates that were studied, only GTP was significantly reactive forming a heterogeneous group of compounds with DL-glyceraldehyde. D-Glucose exhibited no significant reactivity with any of the nucleotide triphosphates, a finding that was supported by UV and fluorescence spectroscopy. Capillary electrophoresis, high-performance liquid chromatography and mass spectrometry allowed for a thorough analysis of the glycated GTP products and demonstrated that the modification of GTP by dl-glyceraldehyde occurred via the classical Amadori pathway.

  17. Adenosine formation and release from neonatal-rat heart cells in culture.

    PubMed Central

    Meghji, P; Holmquist, C A; Newby, A C

    1985-01-01

    The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter. PMID:2996488

  18. Regulation of the calcium release channel from rabbit skeletal muscle by the nucleotides ATP, AMP, IMP and adenosine.

    PubMed

    Laver, D R; Lenz, G K; Lamb, G D

    2001-12-15

    1. Nucleotide activation of skeletal muscle ryanodine receptors (RyRs) was studied in planar lipid bilayers in order to understand RyR regulation in vivo under normal and fatigued conditions. With 'resting' calcium (100 nM cytoplasmic and 1 mM luminal), RyRs had an open probability (P(o)) of approximately 0.01 in the absence of nucleotides and magnesium. ATP reversibly activated RyRs with P(o) at saturation (P(max)) approximately 0.33 and K(a) (concentration for half-maximal activation) approximately 0.36 mM and with a Hill coefficient (n(H)) of approximately 1.8 in RyRs when P(max) < 0.5 and approximately 4 when P(max) > 0.5. 2. AMP was a much weaker agonist (P(max) approximately 0.09) and adenosine was weaker still (P(max) approximately 0.01-0.02), whereas inosine monophosphate (IMP), the normal metabolic end product of ATP hydrolysis, produced no activation at all. 3. Adenosine acted as a competitive antagonist that reversibly inhibited ATP- and AMP-activated RyRs with n(H) approximately 1 and K(i) approximately 0.06 mM at [ATP] < 0.5 mM, increasing 4-fold for each 2-fold increase in [ATP] above 0.5 mM. This is explained by the binding of a single adenosine preventing the cooperative binding of two ATP or AMP molecules, with dissociation constants of 0.4, 0.45 and 0.06 mM for ATP, AMP and adenosine, respectively. Importantly, IMP (< or = 8 mM) had no inhibitory effect whatsoever on ATP-activated RyRs. 4. Mean open (tau(o)) and closed (tau(c)) dwell-times were more closely related to P(o) than to the nucleotide species or individual RyRs. At P(o) < 0.2, RyR regulation occurred via changes in tau(c), whereas at higher P(o) this also occurred via changes in tau(o). The detailed properties of activation and competitive inhibition indicated complex channel behaviour that could be explained in terms of a model involving interactions between different subunits of the RyR homotetramer. 5. The results also show how deleterious adenosine accumulation is to the function of

  19. Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

    PubMed

    Rhaese, H J; Hoch, J A; Groscurth, R

    1977-03-01

    To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

  20. Characterization of adenosine receptors in guinea-pig isolated left atria.

    PubMed Central

    Jahnel, U.; Nawrath, H.

    1989-01-01

    1. The effects of purinergic stimulation on action potential, force of contraction, 86Rb efflux and 45Ca uptake were investigated in guinea-pig left atria. 2. Adenosine exerted a negative inotropic effect which was antagonized by adenosine deaminase but enhanced by dipyridamole. 3. The negative inotropic effect of adenosine was mimicked by 5'-(N-ethyl)-carboxamido-adenosine (NECA) and the isomers of N6-(phenyl-isopropyl)-adenosine, R-PIA and S-PIA. NECA and R-PIA were about 100 times more potent than adenosine, whereas R-PIA was about 100 times more potent than S-PIA. 4. The inotropic effects of adenosine (in the presence of dipyridamole), NECA, R-PIA and S-PIA were competitively antagonized either by theophylline (pA2 about 4.5) or 8-phenyltheophylline (pA2 about 6.3). 5. NECA and R-PIA shortened the action potential duration and increased the rate constant of the efflux of 86Rb in a concentration-dependent manner with no differences in potency; the effects were competitively antagonized by 8-phenyltheophylline. 6. Barium ions reduced the efflux of 86Rb under control conditions and antagonized the increase induced by NECA and R-PIA. 7. NECA and R-PIA significantly reduced 45Ca uptake in beating preparations. 8. It is concluded that adenosine, NECA and R-PIA activate a common receptor population (P1 or A3) on the outside of the cell membrane of atrial heart muscle to increase the potassium conductance and to reduce the action potential and, thereby, calcium influx and force of contraction. PMID:2790380

  1. DNA-Catalyzed Amide Hydrolysis.

    PubMed

    Zhou, Cong; Avins, Joshua L; Klauser, Paul C; Brandsen, Benjamin M; Lee, Yujeong; Silverman, Scott K

    2016-02-24

    DNA catalysts (deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing deoxyribozymes. In one case, the same deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into deoxyribozymes for identifying new catalytic function. The results also suggest the longer-term feasibility of deoxyribozymes as artificial proteases.

  2. Hyperthyroidism modifies ecto-nucleotidase activities in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development.

    PubMed

    Bruno, Alessandra Nejar; Da Silva, Rosane Souza; Bonan, Carla Denise; Battastini, Ana Maria Oliveira; Barreto-chaves, Maria Luiza M; Sarkis, João José Freitas

    2003-11-01

    Here we investigate the possible effects of the hyperthyroidism on the hydrolysis of the ATP to adenosine in the synaptosomes of hippocampus, cerebral cortex and blood serum of rats in different developmental phases. Manifestations of hyperthyroidism include anxiety, nervousness, tachycardia, physical hyperactivity and weight loss amongst others. The thyroid hormones modulate a number of physiological functions in central nervous system, including development, function, expression of adenosine A(1) receptors and transport of neuromodulator adenosine. Thus, hyperthyroidism was induced in male Wistar rats (5-, 60-, 150- and 330-day old) by daily injections of L-thyroxine (T4) for 14 days. Nucleotide hydrolysis was decreased by about 14-52% in both hippocampus and cerebral cortex in 5 to 60-day-old rats. These changes were also observed in rat blood serum. In addition, in 11-month-old rats, inhibition of ADP and AMP hydrolysis persisted in the hippocampus, whereas, in cerebral cortex, an increase in AMP hydrolysis was detected. Thus, hyperthyroidism affects the extracellular nucleotides balance and adenosine production, interfering in neurotransmitter release, development and others physiological processes in different systems.

  3. Progressing batch hydrolysis process

    DOEpatents

    Wright, J.D.

    1985-01-10

    A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.

  4. Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

    PubMed Central

    Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

    2007-01-01

    Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

  5. Metabolic intervention to affect myocardial recovery following ischemia.

    PubMed Central

    Pasque, M K; Wechsler, A S

    1984-01-01

    Myocardial recovery during reperfusion following ischemia is critical to patient survival in a broad spectrum of clinical settings. Myocardial functional recovery following ischemia correlates well with recovery of myocardial adenosine triphosphate (ATP). Adenosine triphosphate recovery is uniformly incomplete during reperfusion following moderate ischemic injury and is therefore subject to manipulation by metabolic intervention. By definition ATP recovery is limited either by (1) energy availability and application in the phosphorylation of adenosine monophosphate (AMP) to ATP or (2) availability of AMP for this conversion. Experimental data suggest that substrate energy and the mechanisms required for its application in the creation of high energy phosphate bonds (AMP conversion to ATP) are more than adequate during reperfusion following moderate ischemic injury. Adenosine monophosphate availability, however, is inadequate following ischemia due to loss of diffusable adenine nucleotide purine metabolites. These purine precursors are necessary to fuel adenine nucleotide salvage pathways. Metabolic interventions that enhance AMP recovery rather than those that improve substrate energy availability during reperfusion are therefore recommended. The mechanisms of various metabolic interventions are discussed in this framework along with the rationale for or against their clinical application. PMID:6428332

  6. Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

    PubMed

    Smith, N; Broadley, K J

    2008-09-01

    Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

  7. Intracoronary Adenosine: Dose-Response Relationship With Hyperemia.

    PubMed

    Adjedj, Julien; Toth, Gabor G; Johnson, Nils P; Pellicano, Mariano; Ferrara, Angela; Floré, Vincent; Di Gioia, Giuseppe; Barbato, Emanuele; Muller, Olivier; De Bruyne, Bernard

    2015-09-01

    The present study sought to establish the dosage of intracoronary (IC) adenosine associated with minimal side effects and above which no further increase in flow can be expected. Despite the widespread adoption of IC adenosine in clinical practice, no wide-ranging, dose-response study has been conducted. A recurring debate still exists regarding its optimal dose. In 30 patients, Doppler-derived flow velocity measurements were obtained in 10 right coronary arteries (RCAs) and 20 left coronary arteries (LCAs) free of stenoses >20% in diameter. Flow velocity was measured at baseline and after 8 ml bolus administrations of arterial blood, saline, contrast medium, and 9 escalating doses of adenosine (4 to 500 μg). The hyperemic value was expressed in percent of the maximum flow velocity reached in a given artery (Q/Qmax, %). Q/Qmax did not increase significantly beyond dosages of 60 μg for the RCA and 160 μg for LCA. Heart rate did not change, whereas mean arterial blood pressure decreased by a maximum of 7% (p < 0.05) after bolus injections of IC adenosine. The incidence of transient A-V blocks was 40% after injection of 100 μg in the RCA and was 15% after injection of 200 μg in the LCA. The duration of the plateau reached 12 ± 13 s after injection of 100 μg in the RCA and 21 ± 6 s after the injection of 200 μg in the LCA. A progressive prolongation of the time needed to return to baseline was observed. Hyperemic response after injection of 8 ml of contrast medium reached 65 ± 36% of that achieved after injection of 200 μg of adenosine. This wide-ranging, dose-response study indicates that an IC adenosine bolus injection of 100 μg in the RCA and 200 μg in the LCA induces maximum hyperemia while being associated with minimal side effects. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  8. Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

    PubMed

    Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel

    2011-06-01

    The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

  9. Delayed production of adenosine underlies temporal modulation of swimming in frog embryo

    PubMed Central

    Dale, Nicholas

    1998-01-01

    To investigate the dynamics of adenosine production in the spinal cord during motor activity, and its possible contribution to the temporal modulation of motor patterns, a sensor sensitive to adenosine at concentrations as low as 10 nm was devised.When pressed against the outside of the spinal cord, the sensor detected slow changes in the levels of adenosine during fictive swimming that ranged from 10 to 650 nm. In four embryos where particularly large signals were recorded due to favourable probe placement, the adenosine levels continued to rise for up to a minute following cessation of activity before slowly returning to baseline. In the remaining thirteen embryos, levels of adenosine started to return slowly to baseline almost immediately after activity had stopped.Inhibitors of adenosine uptake increased the magnitude of the signal recorded and slowed the recovery following cessation of activity.A realistic computational model of the spinal circuitry was combined with models of extracellular breakdown of ATP to adenosine. ATP and adenosine inhibited, as in the real embryo, the voltage-gated K+ and Ca2+ currents, respectively. The model reproduced the temporal run-down of motor activity seen in the real embryo suggesting that synaptic release of ATP together with its extracellular breakdown to adenosine is sufficient to exert time-dependent control over motor pattern generation.The computational analysis also suggested that the delay in the rise of adenosine levels is likely to result from feed-forward inhibition of the 5′-ectonucleotidase in the spinal cord. This inhibition is a key determinant of the rate of run-down. PMID:9679180

  10. Neutrophil-derived 5′-Adenosine Monophosphate Promotes Endothelial Barrier Function via CD73-mediated Conversion to Adenosine and Endothelial A2B Receptor Activation

    PubMed Central

    Lennon, Paul F.; Taylor, Cormac T.; Stahl, Gregory L.; Colgan, Sean P.

    1998-01-01

    During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10−6 M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5′-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5′-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5′-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5′-AMP bioactivity required endothelial CD73-mediated conversion of 5′-AMP to adenosine via its 5′-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5′-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration. PMID:9782120

  11. 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

  12. Analytical Applications of Bioluminescence and Chemiluminescence

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W. (Editor); Picciolo, G. L. (Editor)

    1975-01-01

    Bioluminescence and chemiluminescence studies were used to measure the amount of adenosine triphosphate and therefore the amount of energy available. Firefly luciferase - luciferin enzyme system was emphasized. Photometer designs are also considered.

  13. Rapid method for determination of antimicrobial susceptibilities pattern of urinary bacteria

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

    Method determines bacterial sensitivity to antimicrobial agents by measuring level of adenosine triphosphate remaining in the bacteria. Light emitted during reaction of sample with a mixture of luciferase and luciferin is measured.

  14. 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

  15. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass.

    PubMed

    Davidson, Jesse A; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan; Wischmeyer, Paul E; Klawitter, Jelena

    2016-01-01

    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. 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. 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). Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to

  16. Adenosine transiently modulates stimulated dopamine release in the caudate putamen via A1 receptors

    PubMed Central

    Ross, Ashley E.; Venton, B. Jill

    2014-01-01

    Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 µM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7 %, similar to the 54 ± 6 % decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 minutes. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. PMID:25219576

  17. Evidence for an A2/Ra adenosine receptor in the guinea-pig trachea

    PubMed Central

    Brown, C.M.; Collis, M.G.

    1982-01-01

    1 An attempt was made to determine whether the extracellular adenosine receptor that mediates relaxation in the guinea-pig trachea is of the A1/Ri or A2/Ra subtype. 2 Dose-response curves to adenosine and a number of 5′- and N6-substituted analogues were constructed for the isolated guinea-pig trachea, contracted with carbachol. 3 The 5′-substituted analogues of adenosine were the most potent compounds tested, the order of potency being 5′-N-cyclopropylcarboxamide adenosine (NCPCA) > 5′-N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine > L-N6-phenylisopropyladenosine (L-PIA) > adenosine > D-N6-phenylisopropyladenosine (D-PIA). 4 The difference in potency between the stereoisomers D- and L-PIA on the isolated trachea was at the most five fold. 5 Responses to low doses of adenosine and its analogues were attenuated after treatment with either theophylline or 8-phenyltheophylline. The responses to 2-chloroadenosine were affected to a lesser extent than were those to the other purines. 6 Adenosine transport inhibitors, dipyridamole and dilazep, potentiated responses to adenosine, did not affect those to NCPCA, NECA, L-PIA and D-PIA but significantly reduced the responses to high doses of 2-chloroadenosine. 7 Relaxations evoked by 9-β-D-xylofuranosyladenosine which can activate intracellular but not extracellular adenosine receptors, were attenuated by dipyridamole but unaffected by 8-phenyltheophylline. 8 The results support the existence of an extracellular A2/Ra subtype of adenosine receptor and an intracellular purine-sensitive site, both of which mediate relaxation. PMID:6286021

  18. BCX4430 - A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease.

    PubMed

    Taylor, Raymond; Kotian, Pravin; Warren, Travis; Panchal, Rekha; Bavari, Sina; Julander, Justin; Dobo, Sylvia; Rose, Angela; El-Kattan, Yahya; Taubenheim, Brian; Babu, Yarlagadda; Sheridan, William P

    2016-01-01

    The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. Cellular kinases phosphorylate BCX4430 to a triphosphate that mimics ATP; viral RNA polymerases incorporate the drug's monophosphate nucleotide into the growing RNA chain, causing premature chain termination. BCX4430 is active in vitro against many RNA viral pathogens, including the filoviruses and emerging infectious agents such as MERS-CoV and SARS-CoV. In vivo, BCX4430 is active after intramuscular, intraperitoneal, and oral administration in a variety of experimental infections. In nonclinical studies involving lethal infections with Ebola virus, Marburg virus, Rift Valley fever virus, and Yellow Fever virus, BCX4430 has demonstrated pronounced efficacy. In experiments conducted in several models, both a reduction in the viral load and an improvement in survival were found to be related to the dose of BCX4430. A Phase 1 clinical trial of intramuscular administration of BCX4430 in healthy subjects is currently ongoing. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. All rights reserved.

  19. The 2′,3′-cAMP-adenosine pathway

    PubMed Central

    2011-01-01

    Our recent studies employing HPLC-tandem mass spectrometry to analyze venous perfusate from isolated, perfused kidneys demonstrate that intact kidneys produce and release into the extracellular compartment 2′,3′-cAMP, a positional isomer of the second messenger 3′,5′-cAMP. To our knowledge, this represents the first detection of 2′,3′-cAMP in any cell/tissue/organ/organism. Nuclear magnetic resonance experiments with isolated RNases and experiments in isolated, perfused kidneys suggest that 2′,3′-cAMP likely arises from RNase-mediated transphosphorylation of mRNA. Both in vitro and in vivo kidney experiments demonstrate that extracellular 2′,3′-cAMP is efficiently metabolized to 2′-AMP and 3′-AMP, both of which can be further metabolized to adenosine. This sequence of reactions is called the 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine). Experiments in rat and mouse kidneys show that metabolic poisons increase extracellular levels of 2′,3′-cAMP, 2′-AMP, 3′-AMP, and adenosine; however, little is known regarding the pharmacology of 2′,3′-cAMP, 2′-AMP, and 3′-AMP. What is known is that 2′,3′-cAMP facilitates activation of mitochondrial permeability transition pores, a process that can lead to apoptosis and necrosis, and inhibits proliferation of vascular smooth muscle cells and glomerular mesangial cells. In summary, there is mounting evidence that at least some types of cellular injury, by triggering mRNA degradation, engage the 2′,3′-cAMP-adenosine pathway, and therefore this pathway should be added to the list of biochemical pathways that produce adenosine. Although speculative, it is possible that the 2′,3′-cAMP-adenosine pathway may protect against some forms of acute organ injury, for example acute kidney injury, by both removing an intracellular toxin (2′,3′-cAMP) and increasing an extracellular renoprotectant (adenosine). PMID:21937608

  20. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    PubMed Central

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I.; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits. PMID:29497379

  1. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

    PubMed

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A 2A R present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A 2A R and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A 2A R involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A 2A R-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A 2A R). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

  2. Opiate-induced Changes in Brain Adenosine Levels and Narcotic Drug Responses

    PubMed Central

    Wu, Manhong; Sahbaie, Peyman; Zheng, Ming; Lobato, Robert; Boison, Detlev; Clark, J. David; Peltz, Gary

    2012-01-01

    We have very little information about the metabolomic changes that mediate neurobehavioral responses, including addiction. It was possible that opioid-induced metabolomic changes in brain could mediate some of the pharmacodynamic effects of opioids. To investigate this, opiate-induced brain metabolomic responses were profiled using a semi-targeted method in C57BL/6 and 129Sv1 mice, which exhibit extreme differences in their tendency to become opiate dependent. Escalating morphine doses (10–40 mg/kg) administered over a 4-day period selectively induced a two-fold decrease (p<0.00005) in adenosine abundance in the brainstem of C57BL/6 mice, which exhibited symptoms of narcotic drug dependence; but did not decrease adenosine abundance in 129Sv1 mice, which do not exhibit symptoms of dependence. Based on this finding, the effect of adenosine on dependence was investigated in genetically engineered mice with alterations in adenosine tone in the brain and in pharmacologic experiments. Morphine withdrawal behaviors were significantly diminished (P<0.0004) in genetically engineered mice with reduced adenosine tone in the brainstem, and by treatment with an adenosine receptor1 (A1) agonist (2-chloro-N6-cyclopentyladenosine, 0.5 mg/kg) or an A2a receptor (A2a) antagonist (SCH 58261 1 mg/kg). These results indicate that adenosine homeostasis plays a crucial role in narcotic drug responses. Opiate-induced changes in brain adenosine levels may explain many important neurobehavioral features associated with opiate addiction and withdrawal. PMID:23098802

  3. Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

    PubMed Central

    Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

    2008-01-01

    Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

  4. Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum.

    PubMed

    Cassera, María B; Hazleton, Keith Z; Riegelhaupt, Paul M; Merino, Emilio F; Luo, Minkui; Akabas, Myles H; Schramm, Vern L

    2008-11-21

    Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

  5. Purification and properties of adenosine kinase from rat brain.

    PubMed

    Yamada, Y; Goto, H; Ogasawara, N

    1980-12-04

    Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to apparent homogeneity from rat brain by (NH4)2SO4 fractionation, affinity chromatography on AMP-Sepharose 4B, gel filtration with Sephadex G-100, and DE-52 cellulose column chromatography. The yield was 56% of the initial activity with a final specific activity of 7.8 mumol/min per mg protein. The molecular weight was estimated as 38 000 by gel filtration with Sephadex G-100 and 41 000 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 20% that of adenosine phosphorylation. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad pH optimum at pH 7.5-8.5. The Km value for adenosine was 0.2 microM and the maximum activity was observed at 0.5 microM. At higher concentrations of adenosine, the activity was strongly inhibited. The Km value for ATP was 0.02 mM and that for Mg2+ was 0.1 mM. GTP, dGTP, dATP and UTP were also proved to be effective phosphate donors. Co2+ was as effective as Mg2+, and Ca2+, Mn2+ or Ni2+ showed about 50% of the activity for Mg2+. The kinase is quite unstable, but stable in the presence of a high concentration of salt; e.g., 0.15 M KCl.

  6. Comparative study of cardioprotective effects of uridine-5'-monophosphate and uridine-5'-triphosphate during the early periods of acute myocardial ischemia.

    PubMed

    Bul'on, V V; Krylova, I B; Rodionova, O M; Selina, E N; Evdokimova, N R; Sapronov, N S

    2007-09-01

    In experiments on rats, uridine-5'-monophosphate and uridine-5'-triphosphate reduced the intensity of anaerobic glycolysis and preserved glycogen stores and creatine phosphate balance during the first 60 min after occlusion of the left coronary artery. However, the energy-protective effect of uridine-5'-triphosphate developed 15 min later than the effect of uridine-5'-monophosphate. Uridine-5'-monophosphate, but not uridine-5'-triphosphate, reduced T wave amplitude on ECG and decreased the volume of ischemic injury to the myocardium.

  7. What Do Fats Do in the Body?

    MedlinePlus

    ... the body's main energy source: adenosine triphosphate, or ATP. Arrese works to identify, purify, and determine the ... of Health ( www.nih.gov ), for supplying this article. Measuring Cholesterol Levels Everyone age 20 and older ...

  8. Genetics Home Reference: pyruvate kinase deficiency

    MedlinePlus

    ... glucose is broken down to produce adenosine triphosphate (ATP), the cell's main energy source. PKLR gene mutations ... pyruvate kinase enzyme function, causing a shortage of ATP in red blood cells and increased levels of ...

  9. Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

    PubMed Central

    Haussmann, C; Rohdich, F; Lottspeich, F; Eberhardt, S; Scheuring, J; Mackamul, S; Bacher, A

    1997-01-01

    The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome. PMID:9006053

  10. Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart

    PubMed Central

    Li, Ning; Csepe, Thomas A.; Hansen, Brian J.; Sul, Lidiya V.; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O.; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R.; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul ML; Biesiadecki, Brandon; Hummel, John D; Weiss, Raul; Fedorov, Vadim V.

    2016-01-01

    Background Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor (A1R) and its downstream GIRK channels (IK,Ado). Methods We applied bi-atrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and non-failing human hearts (n=37). Results Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100μM) produced more significant shortening of action potential durations (APD80) in RA (from 290±45ms to 239±41ms, 17.3±10.4%; p<0.01) than LA (from 307±24ms to 286±23ms, 6.7±6.6%; p<0.01). In ten hearts, adenosine induced AF (317±116 sec) that, when sustained (≥2 min), was primarily maintained by one/two localized reentrant drivers in lateral RA. Tertiapin (10–100nM), a selective GIRK channel blocker, counteracted adenosine-induced APD shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher A1R (2.7±1.7 fold; p<0.01) and GIRK4 (1.7±0.8 fold; p<0.05) protein expression than lateral/posterior LA. Conclusions This study revealed a three-fold RA-to-LA A1R protein expression gradient in the human heart, leading to significantly greater RA vs. LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest A1R/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. PMID:27462069

  11. Treatment of out-of-hospital supraventricular tachycardia: adenosine vs verapamil.

    PubMed

    Brady, W J; DeBehnke, D J; Wickman, L L; Lindbeck, G

    1996-06-01

    To compare the use of adenosine and the use of verapamil as out-of-hospital therapy for supraventricular tachycardia (SVT). A period of prospective adenosine use (March 1993 to February 1994) was compared with a historical control period of verapamil use (March 1990 to February 1991) for SVT. Data were obtained for SVT patients treated in a metropolitan, fire-department-based paramedic system serving a population of approximately 1 million persons. Standard drug protocols were used and patient outcomes (i.e., conversion rates, complications, and recurrences) were monitored. During the adenosine treatment period, 105 patients had SVT; 87 (83%) received adenosine, of whom 60 (69%) converted to a sinus rhythm (SR). Vagal maneuvers (VM) resulted in restoration of SR in 8 patients (7.6%). Some patients received adenosine for non-SVT rhythms: 7 sinus tachycardia, 18 atrial fibrilation, 7 wide-complex tachycardia (WCT), and 2 ventricular tachycardia; no non-SVT rhythm converted to SR and none of these patients experienced an adverse effect. Twenty-five patients were hemodynamically unstable (systolic blood pressure < 90 mm Hg), with 20 receiving drug and 13 converting to SR; 8 patients required electrical cardioversion. Four patients experienced adverse effects related to adenosine (chest pain dyspnea, prolonged bradycardia, and ventricular tachycardia). In the verapamil period, 106 patients had SVT: 52 (49%) received verapamil (p < 0.001, compared with the adenosine period), of whom 43 (88%) converted to SR (p = 0.11). Two patients received verapamil for WCT; neither converted to SR and both experienced cardiovascular collapse. VM resulted in restoration of SR in 12 patients (11.0%) (p = 0.52). Sixteen patients were hemodynamically unstable, with 5 receiving drug (p = 0.005) and 5 converting to SR; 9 patients required electrical cardioversion (p = 0.48). Four patients experienced adverse effects related to verapamil (hypotension ventricular tachycardia, ventricular

  12. Esculin hydrolysis by Enterobacteriaceae.

    PubMed

    Edberg, S C; Pittman, S; Singer, J M

    1977-08-01

    Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae.

  13. Esculin hydrolysis by Enterobacteriaceae.

    PubMed Central

    Edberg, S C; Pittman, S; Singer, J M

    1977-01-01

    Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae. PMID:330558

  14. A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

    PubMed Central

    Wilson, Peter M.; LaBonte, Melissa J.; Russell, Jared; Louie, Stan; Ghobrial, Andrew A.; Ladner, Robert D.

    2011-01-01

    Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation. PMID:21576234

  15. Organosolv pretreatment for enzymatic hydrolysis of poplars: I. enzyme hydrolysis of cellulosic residues

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chum, H.L.; Johnson, D.K.; Black, S.

    1988-01-01

    Aspen (Populus tremuloides) and black cottonwood (Populus trichocarpa) organosolv pulps produced in a wide range of solvent composition (between 30 and 70% by volume of methanol) and catalysts (H/sub 2/SO/sub 4/ and H/sub 3/PO/sub 4/) such that the cooking liquor pH less than or equal to 3 are easily digested by enzymes. The total yields of hydrolysis residues (pulps) are in the 40-60% range; the acid-catalyzed delignification followed by enzyme hydrolysis can generate 70-88% of the original six-carbon sugars contained in the wood. Glucomannan and arabinogalactan are dissolved in to the pulping liquor in the pH range of 2-4.5. Lowermore » pH (less than or equal to 3) leads to additional solubilization of six-carbon sugars. These sugars may be fermented directly. From the insoluble hydrolysis residues, 36-41% conversions of wood into fermentable sugars were obtained after enzyme hydrolysis; the starting feedstocks contain 50.8 and 46.6% hexosans, respectively, for aspen and black cottonwood. The kinetics of enzymatic hydrolysis of cellulose can be formally treated as two simultaneous pseudo-first-order reactions in which fast and slow hydrolysis of cellulose occur. Correlations between the glucan digestibility and the effect of the pretreatment have been made. The higher residual xylan content reduces the amount of the rapidly hydrolyzable glucan fraction and lowers the glucan digestibility. The proposed simple kinetic treatment is very helpful in assessing the effect of the pretreatment on pulp enzyme hydrolyzability.« less

  16. Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

    PubMed Central

    Scharbarg, Emeric; Daenens, Marion; Lemaître, Frédéric; Geoffroy, Hélène; Guille-Collignon, Manon; Gallopin, Thierry; Rancillac, Armelle

    2016-01-01

    Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity. PMID:26755200

  17. 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. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Enzymatic saccharification of pretreated wheat straw: comparison of solids-recycling, sequential hydrolysis and batch hydrolysis.

    PubMed

    Pihlajaniemi, Ville; Sipponen, Satu; Sipponen, Mika H; Pastinen, Ossi; Laakso, Simo

    2014-02-01

    In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. The Brain In Vivo Expresses the 2′,3′-cAMP-Adenosine Pathway

    PubMed Central

    Verrier, Jonathan D.; Jackson, Travis C.; Bansal, Rashmi; Kochanek, Patrick M.; Puccio, Ava M.; Okonkwo, David O.; Jackson, Edwin K.

    2012-01-01

    Although multiple biochemical pathways produce adenosine, studies suggest that the 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2′,3′-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2′,3′-cAMP, 3′,5′-cAMP, 2′-AMP, 3′-AMP, or 5′-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2′,3′-cAMP increased 2′-AMP, 3′-AMP and adenosine, and 3′,5′-cAMP increased 5′-AMP and adenosine. In both brain regions, 2′-AMP, 3-AMP and 5′-AMP were converted to adenosine. Microdialysis experiments in 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (TBI; controlled cortical impact model) activated the brain 2,3′-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2′,3′-cAMP to 2′-AMP and to adenosine. In CSF from TBI patients, 2′,3′-cAMP was significantly increased in the initial 12 hours after injury and strongly correlated with CSF levels of 2′-AMP, 3′-AMP, adenosine and inosine. We conclude that in vivo, 2′,3′-cAMP is converted to 2′-AMP/3′-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans. PMID:22360621

  20. Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A2A antagonists MSX-3 and SCH58261, but not the adenosine A1 antagonist DPCPX.

    PubMed

    Collins, Lyndsey E; Galtieri, Daniel J; Brennum, Lise T; Sager, Thomas N; Hockemeyer, Jörg; Müller, Christa E; Hinman, James R; Chrobak, James J; Salamone, John D

    2010-02-01

    Tremulous jaw movements in rats, which can be induced by dopamine (DA) antagonists, DA depletion, and cholinomimetics, have served as a useful model for studies of tremor. Although adenosine A(2A) antagonists can reduce the tremulous jaw movements induced by DA antagonists and DA depletion, there are conflicting reports about the interaction between adenosine antagonists and cholinomimetic drugs. The present studies investigated the ability of adenosine antagonists to reverse the tremorogenic effect of the muscarinic agonist pilocarpine. While the adenosine A(2A) antagonist MSX-3 was incapable of reversing the tremulous jaw movements induced by the 4.0mg/kg dose of pilocarpine, both MSX-3 and the adenosine A(2A) antagonist SCH58261 reversed the tremulous jaw movements elicited by 0.5mg/kg pilocarpine. Systemic administration of the adenosine A(1) antagonist DPCPX failed to reverse the tremulous jaw movements induced by either an acute 0.5mg/kg dose of the cholinomimetic pilocarpine or the DA D2 antagonist pimozide, indicating that the tremorolytic effects of adenosine antagonists may be receptor subtype specific. Behaviorally active doses of MSX-3 and SCH 58261 showed substantial in vivo occupancy of A(2A) receptors, but DPCPX did not. The results of these studies support the use of adenosine A(2A) antagonists for the treatment of tremor. Copyright 2009 Elsevier Inc. All rights reserved.

  1. [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.

  2. A novel mouse model for sudden unexpected death in epilepsy (SUDEP): role of impaired adenosine clearance.

    PubMed

    Shen, Hai-Ying; Li, Tianfu; Boison, Detlev

    2010-03-01

    Sudden unexpected death in epilepsy (SUDEP) is a significant cause of mortality in people with epilepsy. Two postulated causes for SUDEP, cardiac and respiratory depression, can both be explained by overstimulation of adenosine receptors. We hypothesized that SUDEP is a consequence of a surge in adenosine as a result of prolonged seizures combined with deficient adenosine clearance; consequently, blockade of adenosine receptors should prevent SUDEP. Here we induced impaired adenosine clearance in adult mice by pharmacologic inhibition of the adenosine-removing enzymes, adenosine kinase and deaminase. Combination of impaired adenosine clearance with kainic acid-induced seizures triggered sudden death in all animals. Most importantly, the adenosine receptor antagonist caffeine, when given after seizure onset, increased survival from 23.75 +/- 1.35 min to 54.86 +/- 6.59 min (p < 0.01). Our data indicate that SUDEP is due to overactivation of adenosine receptors and that caffeine treatment after seizure onset might be beneficial.

  3. A2B Adenosine Receptor–Mediated Induction of IL-6 Promotes CKD

    PubMed Central

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

    2011-01-01

    Chronic elevation of adenosine, which occurs in the setting of repeated or prolonged tissue injury, can exacerbate cellular dysfunction, suggesting that it may contribute to the pathogenesis of CKD. Here, mice with chronically elevated levels of adenosine, resulting from a deficiency in adenosine deaminase (ADA), developed renal dysfunction and fibrosis. Both the administration of polyethylene glycol–modified ADA to reduce adenosine levels and the inhibition of the A2B adenosine receptor (A2BR) attenuated renal fibrosis and dysfunction. Furthermore, activation of A2BR promoted renal fibrosis in both mice infused with angiotensin II (Ang II) and mice subjected to unilateral ureteral obstruction (UUO). These three mouse models shared a similar profile of profibrotic gene expression in kidney tissue, suggesting that they share similar signaling pathways that lead to renal fibrosis. Finally, both genetic and pharmacologic approaches showed that the inflammatory cytokine IL-6 mediates adenosine-induced renal fibrosis downstream of A2BR. Taken together, these data suggest that A2BR-mediated induction of IL-6 contributes to renal fibrogenesis and shows potential therapeutic targets for CKD. PMID:21511827

  4. Abnormal regulation of adenosine 3′,5′-monophosphate and corticosterone formation in an adrenocortical carcinoma

    PubMed Central

    Ney, R. L.; Hochella, N. J.; Grahame-Smith, D. G.; Dexter, R. N.; Butcher, R. W.

    1969-01-01

    A spontaneously occurring rat adrenocortical carcinoma which produces corticosterone was maintained by transplantation. The carcinoma appeared to utilize corticosterone biosynthetic steps similar to those of the normal adrenal, but the tumor produced only about 1-10% as much corticosterone per unit tissue weight as nontumorous adrenal glands. The tumor demonstrated little or no increase in corticosterone production in response to adrenocorticotropic hormone (ACTH) either in vivo or in vitro. In normal adrenals, ACTH increases the activity of adenyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to adenosine-3′,5′-monophosphate (cyclic AMP), the latter then serving as an intracellular regulator of steroidogenesis. ACTH failed to increase cyclic AMP levels in the tumor in vivo or in slices in vitro, conditions under which there were 50- and 20-fold increases in nontumorous adrenals. However, in homogenates fortified with exogenous ATP, adenyl cyclase activity was comparable in the tumor and adrenals, and cyclic AMP formation was increased 3-fold by ACTH in each. As measured in homogenates, the tumor did not possess a greater ability to destroy cyclic AMP than did normal adrenals. Although ATP levels in the carcinoma were found to be considerably lower than those in normal adrenals, it was not clear that this finding can explain the inability of ACTH to increase cyclic AMP levels in intact tumor cells. While the failure to normally influence cyclic AMP levels in the carcinoma cells could be an important factor in the lack of a steroid response to ACTH, several lines of evidence suggest that the tumor possesses one or more additional abnormalities in the regulation of steroidogenesis. First, in the absence of ACTH stimulation, the tissue concentrations of cyclic AMP were comparable in the tumor and in nontumorous adrenals, but these cyclic AMP levels were associated with a lower level of steroidogenesis in the tumor. Second, tumor slices

  5. Responses of the aorta of the garter snake (Thamnophis sirtalis parietalis) to purines.

    PubMed Central

    Knight, G E; Burnstock, G

    1995-01-01

    1. Isolated aortic rings from the garter snake (Thamnophis sirtalis parietalis) were investigated in order to identify and classify responses to adenosine and adenosine 5'-triphosphate (ATP) and their analogues as part of a comparative study of vertebrate purinoceptors. 2. Adenosine, D-5'-(N-ethylcarboxamide) adenosine (NECA), R- and S-N6-(2-phenylisopropyl) adenosine (R- and S-PIA) and 2-chloroadenosine (2-CA) all concentration-dependently relaxed aorta preconstricted with noradrenaline (NA). The order of potency was: NECA > R-PIA = 2-CA > adenosine > S-PIA. Individual pD2 values for the analogues were: NECA 7.12 +/- 0.13 (9), R-PIA 5.93 +/- 0.25 (7), 2-CA 5.64 +/- 0.40 (5), adenosine 5.04 +/- 0.10 (13) and S-PIA 4.26 +/- 0.10 (7). The order of potency has characteristics of both A1 and A2 receptors and cannot satisfactorily be classified according to the P1-(adenosine) purinoceptor subtypes established in mammalian preparations. 3. ATP, alpha, beta-methylene ATP (alpha, beta-MeATP), 2-methylthio ATP (2MeSATP), beta, gamma-methylene ATP (beta, gamma,-MeATP) and uridine 5'-triphosphate (UTP) all concentration-dependently constricted the isolated aorta. The order of potency was alpha, beta-MeATP = 2MeSATP > ATP > beta, gamma-MeATP > UTP. Only ATP, alpha, beta-MeATP and 2MeSATP consistently produced a maximum response; pD2 values were: ATP 3.98 +/- 0.07 (10), alpha, beta-MeATP 5.86 +/- 0.15 (12) and 2MeSATP 6.06 +/- 0.23 (9). In vessels preconstricted with NA neither ATP nor 2MeSATP caused relaxation in the presence or absence of the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7712027

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

    PubMed

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

    2016-05-01

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

  7. Thyroid hormone action on intermediary metabolism. Part I: respiration, thermogenesis and carbohydrate metabolism.

    PubMed

    Müller, M J; Seitz, H J

    1984-01-02

    The effect of thyroid hormones on mitochondrial respiration are summarized: T3 directly stimulates mitochondrial respiration and the synthesis of adenosine 5'-triphosphate (ATP). Cytosolic ATP availability is increased by a thyroid hormone-induced increase in adenine nucleotide translocation across the mitochondrial membrane; the steady state ATP concentration and the cytosolic ATP/adenosine 5'-diphosphate (ADP) ratio is even decreased in hyperthyroid tissues because of the simultaneous stimulation of the synthesis and consumption of ATP. With regard to the thyroid hormone-induced energy wasting processes, heart work, intra- and interorgan futile cycling and Na+/K+-ATPase are involved to varying degrees. As a consequence of the thyroid hormone-induced hydrolysis of ATP, thermogenesis is increased in hyper- and decreased in hypothyroidism. Despite an increased rate of glucose utilization, clinical and experimental hyperthyroidism is often characterized by an abnormal oral glucose tolerance test. This finding is due to the thyroid hormone-induced increase in intestinal glucose absorption as well as the still enhanced endogenous glucose production in the liver. Hypothyroid patients show a reduced glucose tolerance test because of a decrease in intestinal glucose absorption and a sometimes reduced glucose turnover. The thyroid hormone-induced alterations in glucose metabolism are most probably not due to alterations in serum insulin levels and/or to a peripheral insulin resistance at the receptor level.

  8. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    PubMed

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  9. 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

  10. 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.

  11. 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

  12. 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. © 2015 Japanese Dermatological Association.

  13. EFFECTS OF HYPERTHERMIA AND HYPERTHERMIA PLUS MICROWAVES ON RAT BRAIN ENERGY METABOLISM

    EPA Science Inventory

    The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effects of temperature on adenosine triphosphate concentration (ATP) and creatine phosphate concentration (CP) was determi...

  14. Genetics Home Reference: microphthalmia with linear skin defects syndrome

    MedlinePlus

    ... oxidative phosphorylation , by which mitochondria generate adenosine triphosphate (ATP), the cell's main energy source. It also plays ... Syndrome ClinicalTrials.gov (1 link) ClinicalTrials.gov Scientific Articles on PubMed (1 link) PubMed OMIM (1 link) ...

  15. Progressing batch hydrolysis process

    DOEpatents

    Wright, John D.

    1986-01-01

    A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.

  16. Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury

    NASA Astrophysics Data System (ADS)

    Gaudin, Alice; Yemisci, Müge; Eroglu, Hakan; Lepetre-Mouelhi, Sinda; Turkoglu, Omer Faruk; Dönmez-Demir, Buket; Caban, Seçil; Sargon, Mustafa Fevzi; Garcia-Argote, Sébastien; Pieters, Grégory; Loreau, Olivier; Rousseau, Bernard; Tagit, Oya; Hildebrandt, Niko; Le Dantec, Yannick; Mougin, Julie; Valetti, Sabrina; Chacun, Hélène; Nicolas, Valérie; Desmaële, Didier; Andrieux, Karine; Capan, Yilmaz; Dalkara, Turgay; Couvreur, Patrick

    2014-12-01

    There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

  17. Suppression of Adenosine-Activated Chloride Transport by Ethanol in Airway Epithelia

    PubMed Central

    Raju, Sammeta V.; Wang, Guoshun

    2012-01-01

    Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM) for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (ISC) in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A2B adenosine receptor (A2BAR), largely abolished the adenosine-stimulated chloride transport, suggesting that A2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections. PMID:22442662

  18. Dual specificity and novel structural folding of yeast phosphodiesterase-1 for hydrolysis of second messengers cyclic adenosine and guanosine 3',5'-Monophosphate

    DOE PAGES

    Tian, Yuanyuan; Cui, Wenjun; Huang, Manna; ...

    2014-08-05

    Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a K M of 110 μM and a k cat of 16.9 s⁻¹ for cAMP and a K M of 105 μM and a k cat of 11.8 s₅⁻¹ for cGMP. Thus, the specificity constant (k cat/K McAMP)/(k cat/K M cGMP) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMPmore » at 1.31 Å resolution reveal a new structural folding that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-β-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP.« less

  19. Bacillus anthracis Edema Factor Substrate Specificity: Evidence for New Modes of Action

    PubMed Central

    Göttle, Martin; Dove, Stefan; Seifert, Roland

    2012-01-01

    Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5′-triphosphate, uridine 5′-triphosphate and inosine 5′-triphosphate, in addition to adenosine 5′-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3′:5′-monophosphate, cyclic uridine 3′:5′-monophosphate and cyclic inosine 3′:5′-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed. PMID:22852066

  20. Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine-Releasing Capacity.

    PubMed

    Poppe, Daniel; Doerr, Jonas; Schneider, Marion; Wilkens, Ruven; Steinbeck, Julius A; Ladewig, Julia; Tam, Allison; Paschon, David E; Gregory, Philip D; Reik, Andreas; Müller, Christa E; Koch, Philipp; Brüstle, Oliver

    2018-06-01

    As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system. Stem Cells Translational Medicine 2018;7:477-486. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.