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Sample records for adenosine diphosphate-ribose polymerase

  1. Enhanced poly(adenosine diphosphate ribose) polymerase activity and gene expression in Ewing's sarcoma cells

    SciTech Connect

    Prasad, S.C.; Thraves, P.J.; Bhatia, K.G.; Smulson, M.E.; Dritschilo, A. )

    1990-01-01

    Ewing's sarcoma (ES) is a highly malignant childhood bone tumor and is considered curable by moderate doses of radiotherapy. The addition of chemical inhibitors of the activity of the nuclear enzyme poly(adenosine diphosphate ribose) (poly(ADPR)) polymerase to ES cells in culture results in increased cell killing, a phenomenon called inhibitor sensitization. Since poly(ADPR) polymerase is thought to be associated with DNA repair, it has been suggested that ES cells and other inhibitor-sensitized cells may have a reduced capacity for polymer synthesis resulting in deficient postirradiation recovery. We present here the unexpected observation that in comparison to other cell lines tested, ES cells exhibit a high enzyme activity, higher constitutive levels of the protein, and elevated levels of its mRNA transcript for poly(ADPR) polymerase. No gross amplifications or rearrangements of the gene were observed; however, regulation of poly(ADPR) polymerase in these tumor cells takes place at the level of the gene transcript.

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

    PubMed

    Hemalatha, K; Madhumitha, G

    2016-09-01

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

  3. Cyclic aristeromycin diphosphate ribose: a potent and poorly hydrolysable Ca(2+)-mobilising mimic of cyclic adenosine diphosphate ribose.

    PubMed

    Bailey, V C; Fortt, S M; Summerhill, R J; Galione, A; Potter, B V

    1996-02-01

    Cyclic aristeromycin diphosphate ribose, a carbocyclic analogue of cyclic adenosine diphosphate ribose, was synthesised using a chemo-enzymatic route involving activation of aristeromycin 5'-phosphate by diphenyl phosphochloridate. The calcium-releasing properties of this novel analogue were investigated in sea urchin egg homogenates. While cyclic aristeromycin diphosphate ribose has a calcium release profile similar to that of cyclic adenosine diphosphate ribose (EC50 values are 80 nM and 30 nM, respectively), it is degraded significantly more slowly (t1/2 values are 170 min and 15 min, respectively) and may, therefore, be a useful tool to investigate the activities of cyclic adenosine diphosphate ribose. PMID:8603694

  4. The status of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors in ovarian cancer, part 2: extending the scope beyond olaparib and BRCA1/2 mutations.

    PubMed

    Miller, Rowan E; Ledermann, Jonathan A

    2016-09-01

    Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors have shown clinical activity in epithelial ovarian cancer, leading both the US Food and Drug Administration (FDA) and the European Medicines Agency to approve olaparib for tumors characterized by BRCA1 and BRCA2 mutations. However, it is becoming increasingly evident that tumors that share molecular features with BRCA-mutant tumors-a concept known as BRCAness-also may exhibit defective homologous recombination DNA repair, and therefore will respond to PARP inhibition. A number of strategies have been proposed to identify BRCAness, including identifying defects in other genes that modulate homologous recombination and characterizing the mutational and transcriptional signatures of BRCAness. In addition to olaparib, a number of other PARP inhibitors are in clinical development. This article reviews the development of PARP inhibitors other than olaparib, and discusses the evidence for PARP inhibitors beyond BRCA1/2-mutant ovarian cancer. PMID:27673289

  5. Expression of Poly (Adenosine Diphosphate-Ribose) Polymerase and p53 in Epithelial Ovarian Cancer and Their Role in Prognosis and Disease Outcome

    PubMed Central

    Godoy, Heidi; Mhawech-Fauceglia, Paulette; Beck, Amy; Miller, Austin; Lele, Shashikant; Odunsi, Kunle

    2016-01-01

    Summary PARP, poly (adenosine diphosphate-ribose) polymerase, is a damage-sensing protein, which is essential for the repair of DNA single-strand breaks. PARP and p53 function synergistically in repairing DNA damage and suppressing chromosomal rearrangements. The aim of this study was to determine the expression of PARP and p53 in epithelial ovarian cancer (EOC) and to correlate their expression with clinicopathologic characteristics. PARP and p53 were evaluated using immunohistochemistry applied on a tissue microarray of 189 EOC and their expressions were correlated to clinicopathologic variables, including the age of diagnosis, stage, grade, histologic type, optimal debulking, progression-free survival, and overall survival (OS). PARP and p53 expressions were shown in 61% and 54% of cases, respectively. PARP-positive tumors are more likely to have higher grade (P = 0.03) and complete response to initial first-line chemotherapy (P = 0.009). Patients with positive p53 staining are more likely to be at the advanced stage disease (P = 0.004). Finally, there were no significant associations between PARP and p53 expression and no differences in progression-free survival and OS for PARP or p53 expressions. The overexpression of PARP and p53 in high grade, and advanced stage tumors indicated that these 2 markers might serve as an indicator of aggressive disease behavior. Additional studies are warranted to evaluate the role of PARP and PARP inhibitors in the setting of adjuvant chemotherapy. PMID:21293287

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

    SciTech Connect

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

    2014-02-01

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

  7. Poly(Adenosine 5′-Diphosphate-Ribose) Polymerase Inhibition Counteracts Multiple Manifestations of Experimental Type 1 Diabetic Nephropathy

    PubMed Central

    Drel, Viktor R.; Xu, Weizheng; Zhang, Jie; Pavlov, Ivan A.; Shevalye, Hanna; Slusher, Barbara; Obrosova, Irina G.

    2009-01-01

    This study was aimed at evaluating the role for poly(ADP-ribose) polymerase (PARP) in early nephropathy associated with type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with one of two structurally unrelated PARP inhibitors, 1,5-isoquinolinediol (ISO) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427), at 3 mg/kg−1 · d−1 ip and 30 mg/kg−1 · d−1, respectively, for 10 wk after the first 2 wk without treatment. PARP activity in the renal cortex was assessed by immunohistochemistry and Western blot analysis of poly(ADP-ribosyl)ated proteins. Variables of diabetic nephropathy in urine and renal cortex were evaluated by ELISA, Western blot analysis, immunohistochemistry, and colorimetry. Urinary albumin excretion was increased about 4-fold in diabetic rats, and this increase was prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-associated increase in poly(ADP-ribose) immunoreactivities in renal glomeruli and tubuli and poly(ADP-ribosyl)ated protein level. Renal concentrations of TGF-β1, vascular endothelial growth factor, endothelin-1, TNF-α, monocyte chemoattractant protein-1, lipid peroxidation products, and nitrotyrosine were increased in diabetic rats, and all these changes as well as an increase in urinary TNF-α excretion were completely or partially prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-induced up-regulation of endothelin (B) receptor, podocyte loss, accumulation of collagen-α1 (IY), periodic acid-Schiff-positive substances, fibronectin, and advanced glycation end-products in the renal cortex. In conclusion, PARP activation is implicated in multiple changes characteristic for early nephropathy associated with type 1 diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies. PMID:19854869

  8. Synthesis of DNA and Poly(Adenosine Diphosphate Ribose) in Normal and Chronic Lymphocytic Leukemia Lymphocytes

    PubMed Central

    Berger, Nathan A.; Adams, Jessie W.; Sikorski, Georgina W.; Petzold, Shirley J.; Shearer, William T.

    1978-01-01

    Peripheral blood lymphocytes were isolated from 9 patients with chronic lymphocytic leukemia (CLL) and 12 normal control donors. The cells were assayed for synthesis of DNA and poly-(adenosine diphosphate ribose) (poly[ADPR]) immediately after isolation and on successive days following their treatment with phytohemagglutinin (PHA). Two different techniques were used to measure DNA synthesis. In the standard technique, DNA synthesis was measured by incubating intact cells with [3H]deoxythymidine. In the new technique, the lymphocytes were first rendered permeable to nucleotides, then DNA synthesis was measured by incubating them with [3H]deoxythymidine triphosphate in the presence of deoxyATP, deoxyGTP, deoxyCTP, ATP, and Mg++. Both assays showed the anticipated rise in DNA synthesis after PHA stimulation of normal cells. PHA-stimulated lymphocytes from patients with CLL demonstrated low levels of DNA synthesis in both assay systems. The initial levels of poly(ADPR) synthesis were greater in CLL lymphocytes than in normal cells. Studies with a T-cell-depleted population of normal cells showed the same activity for poly(ADPR) synthesis that was demonstrated by the original population of normal cells. PHA stimulation produced an increase in poly(ADPR) synthesis in both the normal and CLL cells. The increase in poly(ADPR) synthesis in normal cells was coincident with the increase in DNA synthesis. The increase in poly(ADPR) synthesis in the CLL cells was dissociated from the delayed and diminished increase in DNA synthesis. Thus, CLL cells have higher than normal initial levels of poly(ADPR) synthesis. Poly(ADPR) synthesis is dissociated from DNA synthesis in CLL cells whereas it varies directly with DNA synthesis in normal lymphocytes. PMID:659624

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

    PubMed Central

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

    2016-01-01

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

  10. Synthesis of cyclic adenosine 5′-diphosphate ribose analogues: a C2′ endo/syn “southern” ribose conformation underlies activity at the sea urchin cADPR receptor† †This paper is part of an Organic & Biomolecular Chemistry web theme issue on chemical biology.

    PubMed Central

    Moreau, Christelle; Ashamu, Gloria A.; Bailey, Victoria C.; Galione, Antony; Guse, Andreas H.

    2011-01-01

    Novel 8-substituted base and sugar-modified analogues of the Ca2+ mobilizing second messenger cyclic adenosine 5′-diphosphate ribose (cADPR) were synthesized using a chemoenzymatic approach and evaluated for activity in sea urchin egg homogenate (SUH) and in Jurkat T-lymphocytes; conformational analysis investigated by 1H NMR spectroscopy revealed that a C2′ endo/syn conformation of the “southern” ribose is crucial for agonist or antagonist activity at the SUH-, but not at the T cell-cADPR receptor. PMID:20976353

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

    PubMed Central

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

    1969-01-01

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

  12. 2'-deoxy cyclic adenosine 5'-diphosphate ribose derivatives: importance of the 2'-hydroxyl motif for the antagonistic activity of 8-substituted cADPR derivatives.

    PubMed

    Zhang, Bo; Wagner, Gerd K; Weber, Karin; Garnham, Clive; Morgan, Anthony J; Galione, Antony; Guse, Andreas H; Potter, Barry V L

    2008-03-27

    The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues. PMID:18303825

  13. NAD⁺ in aging, metabolism, and neurodegeneration.

    PubMed

    Verdin, Eric

    2015-12-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a coenzyme found in all living cells. It serves both as a critical coenzyme for enzymes that fuel reduction-oxidation reactions, carrying electrons from one reaction to another, and as a cosubstrate for other enzymes such as the sirtuins and poly(adenosine diphosphate-ribose) polymerases. Cellular NAD(+) concentrations change during aging, and modulation of NAD(+) usage or production can prolong both health span and life span. Here we review factors that regulate NAD(+) and discuss how supplementation with NAD(+) precursors may represent a new therapeutic opportunity for aging and its associated disorders, particularly neurodegenerative diseases. PMID:26785480

  14. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

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

  15. 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

  16. Adenosine and Sleep

    PubMed Central

    Bjorness, Theresa E; Greene, Robert W

    2009-01-01

    Over the last several decades the idea that adenosine (Ado) plays a role in sleep control was postulated due in large part to pharmacological studies that showed the ability of Ado agonists to induce sleep and Ado antagonists to decrease sleep. A second wave of research involving in vitro cellular analytic approaches and subsequently, the use of neurochemical tools such as microdialysis, identified a population of cells within the brainstem and basal forebrain arousal centers, with activity that is both tightly coupled to thalamocortical activation and under tonic inhibitory control by Ado. Most recently, genetic tools have been used to show that Ado receptors regulate a key aspect of sleep, the slow wave activity expressed during slow wave sleep. This review will briefly introduce some of the phenomenology of sleep and then summarize the effect of Ado levels on sleep, the effect of sleep on Ado levels, and recent experiments using mutant mouse models to characterize the role for Ado in sleep control and end with a discussion of which Ado receptors are involved in such control. When taken together, these various experiments suggest that while Ado does play a role in sleep control, it is a specific role with specific functional implications and it is one of many neurotransmitters and neuromodulators affecting the complex behavior of sleep. Finally, since the majority of adenosine-related experiments in the sleep field have focused on SWS, this review will focus largely on SWS; however, the role of adenosine in REM sleep behavior will be addressed. PMID:20190965

  17. Rat cardiac myocyte adenosine transport and metabolism

    SciTech Connect

    Ford, D.A.; Rovetto, M.J.

    1987-01-01

    Based on the importance of myocardial adenosine and adenine nucleotide metabolism, the adenosine salvage pathway in ventricular myocytes was studied. Accurate estimates of transport rates, separate from metabolic fllux, were determined. Adenosine influx was constant between 3 and 60 s. Adenosine metabolism maintained intracellular adenosine concentrations < 10% of the extracellular adenosine concentrations and thus unidirectional influx could be measured. Myocytes transported adenosine via saturable and nonsaturable processes. A minimum estimate of the V/sub max/ of myocytic adenosine kinase indicated the saturable component of adenosine influx was independent of adenosine kinase activity. Saturable transport was inhibited by nitrobenzylthioinosine and verapamil. Extracellular adenosine taken up myocytes was rapidly phosphorylated to adenine taken up by myocytes was rapidly phosphorylated to adenine nucleotides. Not all extracellular adenosine, though, was phosphorylated on entering myocytes, since free, as opposed to protein-bound, intracellular adenosine was detected after digitonin extraction of cells in the presence of 1 mM ethylene-diaminetetraacetic acid.

  18. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

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

  19. Nicotinamide in dermatology and photoprotection.

    PubMed

    Surjana, Devita; Damian, Diona L

    2011-01-01

    Nicotinamide (the amide form of vitamin B3) has been used in dermatology for more than 40 years for a diverse range of conditions including acne, rosacea, autoimmune bullous dermatoses, and now the treatment and prevention of photoaging and photoimmunosuppression. The broad clinical effects of nicotinamide may be explained by its role as a cellular energy precursor, a modulator of inflammatory cytokines, and an inhibitor of the nuclear enzyme poly(adenosine diphosphate-ribose) polymerase-1, which plays a significant role in DNA repair, maintenance ofgenomic stability, and cellular response to injury including inflammation and apoptosis. This review outlines the use of nicotinamide for inflammatory dermatoses and photoaging and focuses on its emerging role in photoprotection.

  20. Glaucocalyxin A inhibits the growth of liver cancer Focus and SMMC-7721 cells

    PubMed Central

    TANG, LISHA; JIN, XIAOFENG; HU, XIAOHUI; HU, XIAODING; LIU, ZULONG; YU, LONG

    2016-01-01

    Liver cancer is one of the most common types of cancer, and hepatoma demonstrates a poor long-term prognosis. The present study reports that glaucocalyxin A (GLA), a natural product isolated from Rabdosia umbrosa, inhibits the growth of the liver cancer Focus and SMMC-7721 cell lines in a dose- and time-dependent manner. The present study revealed that GLA arrested the liver cancer cells at the G2/M stage of the cell cycle and led to decreased expression of caspase 3 and the cleavage of poly(adenosine diphosphate-ribose) polymerase. Overall, the present study demonstrated that GLA inhibits the growth of liver cancer cells by G2/M stage cell-cycle arrest and cell apoptosis. PMID:26893714

  1. New Strategies for Multimodality Therapy in Treating Locally Advanced Cervix Cancer.

    PubMed

    Verma, Jonathan; Monk, Bradley J; Wolfson, Aaron H

    2016-10-01

    Cervical cancer is the fourth most common cause of cancer of women worldwide. In the developing world, it comprises 12% of all cancers of women. Since 1999, the mainstay of treatment for locally advanced cervical cancer (LACC) has been concurrent cisplatin-based chemoradiation. However, outcomes in this disease remain suboptimal, with long-term progression-free survival and overall survival rates of approximately 60%. There are several new strategies of combined modality treatment under evaluation in LACC, including chemotherapy before and after treatment as well as novel agents such as poly-adenosine diphosphate ribose polymerase inhibitors, antiangiogenic blockage, and immunotherapy. We provide a brief overview of these strategies and their potential in the treatment of women with LACC. PMID:27619255

  2. 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. PMID:27638696

  3. 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.

  4. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

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

    1976-01-01

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

  5. 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

  6. 5'-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart.

    PubMed

    Headrick, J P; Peart, J; Hack, B; Garnham, B; Matherne, G P

    2001-11-01

    We examined myocardial 5'-adenosine monophosphate (5'-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. MVO(2) increased from 71+/-8 microl O(2)/min per g in guinea pig to 138+/-17 and 221+/-15 microl O(2)/min per g in rat and mouse. VO(2)/beat was 0.42+/-0.03, 0.50+/-0.03 and 0.55+/-0.04 microl O(2)/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca(2+) sensitivity declined as heart mass increased. Net myocardial 5'-AMP dephosphorylation increased significantly as mass declined (3.8+/-0.5, 9.0+/-1.4 and 11.0+/-1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5'-AMP catabolism, coronary venous [adenosine] was similar in guinea pig, rat and mouse (45+/-8, 69+/-10 and 57+/-14 nM, respectively). Comparable venous [adenosine] was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35-45% of inosine and its catabolites derive from 5'-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A(1) receptor sensitivity being greatest in mouse while A(2) sensitivity was greatest in guinea pig. In summary: (i) cardiac 5'-AMP dephosphorylation, VO(2), contractility and Ca(2+) sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A(2) sensitivity relative to A(1) sensitivity in larger hearts.

  7. Inhibition of Influenza Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate

    PubMed Central

    Eriksson, Bertil; Helgstrand, Erik; Johansson, Nils Gunnar; Larsson, Alf; Misiorny, Alfons; Noren, Jan Olof; Philipson, Lennart; Stenberg, Kjell; Stening, Goran; Stridh, Stig; Öberg, Bo

    1977-01-01

    Ribavirin 5′-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5′-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5′-triphosphate and guanosine 5′-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP. PMID:879760

  8. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    MedlinePlus

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

  9. Features of adenosine metabolism of mouse heart.

    PubMed

    Deussen, Andreas; Weichsel, Johannes; Pexa, Annette

    2006-11-01

    Adenosine metabolism and transport were evaluated in the isolated perfused mouse heart and compared with the well-established model of isolated perfused guinea pig heart. Coronary venous release of adenosine under well-oxygenated conditions in the mouse exceeds that in the guinea pig threefold when related to tissue mass. Total myocardial adenosine production rate under this condition was approximately 2 nmol/min per gramme and similar in both species. Coronary resistance vessels of mice are highly sensitive to exogenous adenosine, and the threshold for adenosine-induced vasodilation is approximately 30 nmol/l. Adenosine membrane transport was largely insensitive to nitrobenzyl-thioinosine (NBTI) in mouse heart, which is in contrast to guinea pig and several other species. This indicates the dominance of NBTI-insensitive transporters in mouse heart. For future studies, the assessment of cytosolic and extracellular adenosine metabolism and its relationship with coronary flow will require the use of more effective membrane transport blockers.

  10. Fluorescent ligands for adenosine receptors.

    PubMed

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

    2013-01-01

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

  11. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  12. Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor.

    PubMed

    Kobayashi, S; Conforti, L; Pun, R Y; Millhorn, D E

    1998-04-01

    1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in

  13. Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor

    PubMed Central

    Kobayashi, Shuichi; Conforti, Laura; Pun, Raymund Y K; Millhorn, David E

    1998-01-01

    The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6–22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and

  14. Effects of adenosine on polymorphonuclear leucocyte function, cyclic 3': 5'-adenosine monophosphate, and intracellular calcium.

    PubMed Central

    Nielson, C. P.; Vestal, R. E.

    1989-01-01

    1. Inhibition of human polymorphonuclear leucocyte (PMN) function by adenosine was studied with respect to effects of adenosine on intracellular cyclic AMP and calcium during the PMN respiratory burst. 2. The adenosine analogue 5'-N-ethylcarboxamide-adenosine (NECA) and L-N6-phenyl-isopropyl-adenosine (L-PIA) inhibited PMN oxygen metabolite generation with relative potencies (NECA greater than adenosine greater than L-PIA) characteristic of an A2 receptor. 3. The respiratory burst was inhibited by adenosine when PMN were activated by calcium ionophore or chemotactic peptide but not when cells where activated by oleoyl-acetyl-glycerol (OAG). 4. Adenosine increased intracellular cyclic AMP during the PMN respiratory burst regardless of whether cells were stimulated by ionophore, chemotactic peptide or OAG. 5. To determine whether the differences in cell inhibition by adenosine were related to differences in intracellular calcium mobilization by each activating agent, calcium was evaluated with the fluorescent probe, indo-1. Adenosine suppressed the increase in intracellular calcium following PMN activation by calcium ionophore or chemotactic peptide. In contrast, calcium did not increase in PMN activated by OAG and adenosine did not affect intracellular calcium changes following this stimulus. 6. These results demonstrate that physiological concentrations of adenosine inhibit the PMN respiratory burst in association with an increase in intracellular cyclic AMP and reduction of intracellular calcium. PMID:2547490

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

    SciTech Connect

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

    1990-04-01

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

  16. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  17. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  18. Regulation of Cardiovascular Development by Adenosine and Adenosine-Mediated Embryo Protection

    PubMed Central

    Rivkees, Scott A.; Wendler, Christopher C.

    2012-01-01

    Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methlyxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (ARs). We examined how adenosine acts via A1ARs to influence embryo development. Transgenic mice were studied along with embryo cultures. Embryos lacking A1ARs were markedly growth retarded following intrauterine hypoxia exposure. Studies of mice selectively lacking A1AR in the heart identify the heart as a key site of adenosines embryo protective effects. Studies of isolated embryos showed that adenosine plays a key role in modulating embryo cardiac function, especially in the setting of hypoxia. When pregnant mice were treated during embryogenesis with the adenosine antagonist caffeine, adult mice had abnormal heart function. Adenosine acts via A1ARs to play an essential role in protecting the embryo against intra uterine stress, and adenosine antagonists, including caffeine, may be an unwelcome exposure for the embryo. PMID:22423036

  19. Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

    PubMed

    Zheng, G Z; Lee, C; Pratt, J K; Perner, R J; Jiang, M Q; Gomtsyan, A; Matulenko, M A; Mao, Y; Koenig, J R; Kim, K H; Muchmore, S; Yu, H; Kohlhaas, K; Alexander, K M; McGaraughty, S; Chu, K L; Wismer, C T; Mikusa, J; Jarvis, M F; Marsh, K; Kowaluk, E A; Bhagwat, S S; Stewart, A O

    2001-08-20

    A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

  20. 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.

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

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  2. Optical Aptasensors for Adenosine Triphosphate

    PubMed Central

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

    2016-01-01

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

  3. 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

  4. The adenosine neuromodulation system in schizophrenia.

    PubMed

    Rial, Daniel; Lara, Diogo R; Cunha, Rodrigo A

    2014-01-01

    The management of schizophrenia endophenotypes, namely positive, negative, and cognitive symptoms is still an open goal, justifying the search of novel therapeutic avenues. We now review the evidence supporting the interest in targeting the adenosine modulation system to counteract the core features of schizophrenia. This interest is forwarded by the combined ability of strategies aimed at bolstering adenosine levels together with the increasingly recognized impact of adenosine A2A receptors to control dopaminergic signaling, working memory, and behavioral sensitization; this is further heralded by the suggested clinical effectiveness of therapies increasing extracellular adenosine such as dipyridamole and allopurinol and the emergent recognition of a role for adenosine in neurodevelopment. Finally, the combined role of A1 and A2A receptors in assisting the implementation of adaptive changes and encoding of information salience in neuronal circuits together with the adaptive alterations of A1 and A2A receptor density upon brain dysfunction prompts the novel working hypothesis that the parallel imbalance of adenosine formation and of A1 and A2A receptors blurs the adequate encoding of information salience in neuronal circuits, which we propose to be a core pathogenic feature in the development of schizophrenia endophenotypes. This proposal should also provide a rationale to assist the design of future therapeutic intervention targeting the adenosine modulation system to manage schizophrenia endophenotypes: these should not be based only on an attempt to target adenosine kinase-A1 receptors or only A2A receptors, but should instead simultaneously target these two arms of the adenosine modulation system. PMID:25175974

  5. DNA polymerase profiling.

    PubMed

    Summerer, Daniel

    2008-01-01

    We report a simple homogeneous fluorescence assay for quantification of DNA polymerase function in high throughput. The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon extension of the template strand. A resulting distance alteration is reported by fluorescence resonance energy transfer between two dyes introduced into the molecular beacon stem. We describe real-time reaction profiling of two model DNA polymerases. We demonstrate kinetic characterization, rapid optimization of reaction conditions, and inhibitor profiling using the presented assay. Furthermore, to supersede purification steps in screening procedures of DNA polymerase mutant libraries, detection of enzymatic activity in bacterial expression lysates is described.

  6. Cloning and expression of an A1 adenosine receptor from rat brain

    SciTech Connect

    Mahan, L.C.; McVittie, L.D.; Smyk-Randall, E.M.; Nakata, H.; Monsma, F.J. Jr.; Gerfen, C.R.; Sibley, D.R. )

    1991-07-01

    The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.

  7. Adenosine receptors as drug targets — what are the challenges?

    PubMed Central

    Chen, Jiang-Fan; Eltzschig, Holger K.; Fredholm, Bertil B.

    2014-01-01

    Adenosine signalling has long been a target for drug development, with adenosine itself or its derivatives being used clinically since the 1940s. In addition, methylxanthines such as caffeine have profound biological effects as antagonists at adenosine receptors. Moreover, drugs such as dipyridamole and methotrexate act by enhancing the activation of adenosine receptors. There is strong evidence that adenosine has a functional role in many diseases, and several pharmacological compounds specifically targeting individual adenosine receptors — either directly or indirectly — have now entered the clinic. However, only one adenosine receptor-specific agent — the adenosine A2A receptor agonist regadenoson (Lexiscan; Astellas Pharma) — has so far gained approval from the US Food and Drug Administration (FDA). Here, we focus on the biology of adenosine signalling to identify hurdles in the development of additional pharmacological compounds targeting adenosine receptors and discuss strategies to overcome these challenges. PMID:23535933

  8. Mast Cell Adenosine Receptors Function: A Focus on the A3 Adenosine Receptor and Inflammation

    PubMed Central

    Rudich, Noam; Ravid, Katya; Sagi-Eisenberg, Ronit

    2012-01-01

    Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells (MCs), as an attractive drug candidate. Four subtypes (A1, A2a, A2b, and A3) of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R) in mediating hyper responsiveness to adenosine in MCs, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human MCs. The relevance of mouse studies to the human is discussed. PMID:22675325

  9. [Adenosine deaminase in experimental trypanosomiasis: future implications].

    PubMed

    Pérez-Aguilar, Mary Carmen; Rondón-Mercado, Rocío

    2015-09-01

    The adenosine deaminase represents a control point in the regulation of extracellular adenosine levels, thus playing a critical role in the modulation of purinergic responses to certain pathophysiological events. Several studies have shown that serum and plasma enzyme levels are elevated in some diseases caused by microorganisms, which may represent a compensatory mechanism due to the elevated levels of adenosine and the release of inflammatory mediators. Recent research indicates that adenosine deaminase activity decreases and affects hematological parameters of infected animals with Trypanosoma evansi, so that such alterations could have implications in the pathogenesis of the disease. In addition, the enzyme has been detected in this parasite; allowing the inference that it could be associated with the vital functions of the same, similar to what occurs in mammals. This knowledge may be useful in the association of chemotherapy with specific inhibitors of the enzyme in future studies.

  10. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

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

    1991-01-01

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

  11. Adenosine inhibits glutamatergic input to basal forebrain cholinergic neurons

    PubMed Central

    Hawryluk, J. M.; Ferrari, L. L.; Keating, S. A.

    2012-01-01

    Adenosine has been proposed as an endogenous homeostatic sleep factor that accumulates during waking and inhibits wake-active neurons to promote sleep. It has been specifically hypothesized that adenosine decreases wakefulness and promotes sleep recovery by directly inhibiting wake-active neurons of the basal forebrain (BF), particularly BF cholinergic neurons. We previously showed that adenosine directly inhibits BF cholinergic neurons. Here, we investigated 1) how adenosine modulates glutamatergic input to BF cholinergic neurons and 2) how adenosine uptake and adenosine metabolism are involved in regulating extracellular levels of adenosine. Our experiments were conducted using whole cell patch-clamp recordings in mouse brain slices. We found that in BF cholinergic neurons, adenosine reduced the amplitude of AMPA-mediated evoked glutamatergic excitatory postsynaptic currents (EPSCs) and decreased the frequency of spontaneous and miniature EPSCs through presynaptic A1 receptors. Thus we have demonstrated that in addition to directly inhibiting BF cholinergic neurons, adenosine depresses excitatory inputs to these neurons. It is therefore possible that both direct and indirect inhibition may synergistically contribute to the sleep-promoting effects of adenosine in the BF. We also found that blocking the influx of adenosine through the equilibrative nucleoside transporters or inhibiting adenosine kinase and adenosine deaminase increased endogenous adenosine inhibitory tone, suggesting a possible mechanism through which adenosine extracellular levels in the basal forebrain are regulated. PMID:22357797

  12. RNA Polymerases of Maize: Nuclear RNA Polymerases*

    PubMed Central

    Strain, Gustave C.; Mullinix, Kathleen P.; Bogorad, Lawrence

    1971-01-01

    Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The two enzymes can be separated on DEAE-cellulose columns. Enzymes I and II are eluted with 0.08 and 0.20 M (NH4)2SO4, respectively. Both enzymes prefer maize nuclear DNA as a template; they are also more active in the presence of Mg++ than Mn++ and are inhibited by (NH4)2-SO4 or KCl. Neither enzyme is inhibited by rifamycin SV. Enzyme II is strongly inhibited by α-amanitin, whereas enzyme I is not significantly affected. Their ability to use native and denatured DNA as templates varies according to the extent and method of purification of the polymerase. Furthermore, enzyme II can be resolved by DEAE-chromatography or glycerol-gradient centrifugation into two components, one of which prefers native DNA, while the other prefers denatured DNA. PMID:5288239

  13. Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved.

    PubMed

    Vacas, Javier; Fernández, Mercedes; Ros, Manuel; Blanco, Pablo

    2003-12-01

    Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.

  14. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium

    SciTech Connect

    Pratt, A.D.; Clancy, G.; Welsh, M.J.

    1986-08-01

    Adenosine is a local regulator of a variety of physiological functions in many tissues and has been observed to stimulate secretion in several Cl-secreting epithelia. In canine tracheal epithelium the authors found that adenosine stimulates Cl secretion from both the mucosal and submucosal surfaces. Addition of adenosine, or its analogue 2-chloroadenosine, to the mucosal surface potently stimulated Cl secretion with no effect on the rate of Na absorption. Stimulation resulted from an interaction of adenosine with adenosine receptors, because it was blocked by the adenosine receptor blocker, 8-phenyltheophylline. The adenosine receptor was a stimulatory receptor as judged by the rank-order potency of adenosine and its analogues and by the increase in cellular adenosine 3',5'-cyclic monophosphate levels produced by 2-chloroadenosine. Adenosine also stimulated Cl secretion when it was added to the submucosal surface, although the maximal increase in secretion was less and it was much less potent. The observation that mucosal 8-phenyletheophylline blocked the effect of submucosal 2-chloroadenosine, whereas submucosal 8-phenyltheophylline did not prevent a response to mucosal or submucosal 2-chloroadenosine, suggests that adenosine receptors are located on the mucosal surface. Thus submucosal adenosine may stimulate secretion by crossing the epithelium and interacting with receptors located on the mucosal surface. Because adenosine can be released from mast cells located in the airway lumen in response to inhaled material, and because adenosine stimulated secretion from the mucosal surface, it may be in a unique position to control the epithelium on a regional level.

  15. Pelargonium quercetorum Agnew induces apoptosis without PARP or cytokeratin 18 cleavage in non-small cell lung cancer cell lines

    PubMed Central

    Aztopal, Nazlihan; Cevatemre, Buse; Sarimahmut, Mehmet; Ari, Ferda; Dere, Egemen; Ozel, Mustafa Zafer; Firat, Mehmet; Ulukaya, Engin

    2016-01-01

    Pelargonium species have various uses in folk medicine as traditional remedies, and several of them have been screened for their biological activity, including anticancer. Pelargonium quercetorum Agnew (P. quercetorum) is traditionally used for its anthelminthic activity. However, little is known about its biological activity or its effect on cancer cells. The aim of the present study was to determine the cytotoxic activity of P. quercetorum extract on lung cancer cell lines with varying properties. Following the analyses of its chemical composition, the cytotoxic activity was screened by the adenosine triphosphate viability test. M30-Apoptosense® and M65 EpiDeath® enzyme-linked immunosorbent assays were used to determine the cell death mode (apoptosis vs. necrosis). For apoptosis, additional methods, including Annexin-V-fluorescein isothiocyanate (FITC) and Hoechst 33342 staining, were employed. The cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) was assayed by western blotting to further dissect the apoptosis mechanism. The methanol extract of P. quercetorum caused cytotoxic activity in a dose-dependent manner. The mode of cell death was apoptosis, as evidenced by the positive staining of the cells for Annexin-V-FITC and the presence of pyknotic nuclei. Notably, neither PARP cleavage nor cytokeratin 18 fragmentation were observed. P.quercetorum caused cell death by an apoptosis mechanism that is slightly different from classical apoptosis. Therefore, future in vivo experiments are required for further understanding of the effect of this plant on cancer cells. PMID:27446448

  16. The expanding polymerase universe.

    PubMed

    Goodman, M F; Tippin, B

    2000-11-01

    Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

  17. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

    Jacobson, Kenneth A.; Ukena, Dieter; Padgett, William; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki ~ 10−9 −10−8 M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed. PMID:3036153

  18. Determination of adenosine effects and adenosine receptors in murine corpus cavernosum.

    PubMed

    Tostes, Rita C; Giachini, Fernanda R C; Carneiro, Fernando S; Leite, Romulo; Inscho, Edward W; Webb, R Clinton

    2007-08-01

    This study tested the hypothesis that adenosine, in murine corpora cavernosa, produces direct relaxation of smooth muscle cells and inhibition of contractile responses mediated by sympathetic nerve stimulation. Penes were excised from anesthetized male C57BL/6 mice, dissected, and cavernosal strips were mounted to record isometric force. Adenosine, 2-chloroadenosine (stable analog of adenosine), and 2-phenylaminoadenosine (CV1808) (A2(A)/A2(B) agonist) produced concentration-dependent relaxations of phenylephrine-contracted tissues. Relaxation to 2-chloroadenosine was inhibited, in a concentration-dependent manner, by 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH58261; A2(A) antagonist; 10(-9)-10(-6) M) and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamida (MRS1706; A2(B) antagonist; 10(-8)-10(-6) M). The combination of both antagonists abrogated 2-chloroadenosine-induced relaxation. Electrical field stimulation (EFS; 1-32 Hz) of adrenergic nerves produced frequency-dependent contractions that were inhibited by compounds that increase adenosine levels, such as 5'-iodotubercidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor), and dipyridamole (inhibitor of adenosine transport). The adenosine A1 receptor agonist N(6)-cyclopentyladenosine (C8031) right-shifted contractile responses to EFS, with a significant inhibitory effect at 10(-6) M. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine (C101) (10(-7) M) enhanced contractile responses to EFS and eliminated the inhibitory effects of 5'-iodotubercidin. Dipyridamole and 5'-iodotubercidin had no effect on adenosine-mediated relaxation. In summary, adenosine directly relaxes cavernosal smooth muscle cells, by the activation of A2(A)/A2(B) receptor subtypes. In addition, adenosine negatively modulates sympathetic neurotransmission, by A1 receptor

  19. Working memory and the homeostatic control of brain adenosine by adenosine kinase.

    PubMed

    Singer, P; McGarrity, S; Shen, H-Y; Boison, D; Yee, B K

    2012-06-28

    The neuromodulator adenosine maintains brain homeostasis and regulates complex behaviour via activation of inhibitory and excitatory adenosine receptors (ARs) in a brain region-specific manner. AR antagonists such as caffeine have been shown to ameliorate cognitive impairments in animal disease models but their effects on learning and memory in normal animals are equivocal. An alternative approach to reduce AR activation is to lower the extracellular tone of adenosine, which can be achieved by up-regulating adenosine kinase (ADK), the key enzyme of metabolic adenosine clearance. However, mice that globally over-express an Adk transgene ('Adk-tg' mice) were devoid of a caffeine-like pro-cognitive profile; they instead exhibited severe spatial memory deficits. This may be mechanistically linked to cortical/hippocampal N-methyl-d-aspartate receptor (NMDAR) hypofunction because the motor response to acute MK-801 was also potentiated in Adk-tg mice. Here, we evaluated the extent to which the behavioural phenotypes of Adk-tg mice might be modifiable by up-regulating adenosine levels in the cortex/hippocampus. To this end, we investigated mutant 'fb-Adk-def' mice in which ADK expression was specifically reduced in the telencephalon leading to a selective increase in cortical/hippocampal adenosine, while the rest of the brain remained as adenosine-deficient as in Adk-tg mice. The fb-Adk-def mice showed an even greater impairment in spatial working memory and a more pronounced motor response to NMDAR blockade than Adk-tg mice. These outcomes suggest that maintenance of cortical/hippocampal adenosine homeostasis is essential for effective spatial memory and deviation in either direction is detrimental with increased expression seemingly more disruptive than decreased expression.

  20. 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

  1. Metabolomic analysis of exercise effects in the POLG mitochondrial DNA mutator mouse brain.

    PubMed

    Clark-Matott, Joanne; Saleem, Ayesha; Dai, Ying; Shurubor, Yevgeniya; Ma, Xiaoxing; Safdar, Adeel; Beal, Myron Flint; Tarnopolsky, Mark; Simon, David K

    2015-11-01

    Mitochondrial DNA (mtDNA) mutator mice express a mutated form of mtDNA polymerase gamma that results an accelerated accumulation of somatic mtDNA mutations in association with a premature aging phenotype. An exploratory metabolomic analysis of cortical metabolites in sedentary and exercised mtDNA mutator mice and wild-type littermate controls at 9-10 months of age was performed. Pathway analysis revealed deficits in the neurotransmitters acetylcholine, glutamate, and aspartate that were ameliorated by exercise. Nicotinamide adenine dinucleotide (NAD) depletion and evidence of increased poly(adenosine diphosphate-ribose) polymerase 1 (PARP1)activity were apparent in sedentary mtDNA mutator mouse cortex, along with deficits in carnitine metabolites and an upregulated antioxidant response that largely normalized with exercise. These data highlight specific pathways that are altered in the brain in association with an accelerated age-related accumulation of somatic mtDNA mutations. These results may have relevance to age-related neurodegenerative diseases associated with mitochondrial dysfunction, such as Alzheimer's disease and Parkinson's disease and provide insights into potential mechanisms of beneficial effects of exercise on brain function.

  2. Adenosine induced coronary spasm – A rare presentation

    PubMed Central

    Arora, P.; Bhatia, V.; Arora, M.; Kaul, U.

    2014-01-01

    Adenosine is commonly used as a pharmacological agent in myocardial perfusion imaging, as an antiarrhythmic agent, and in Cath Lab. during PCI for treating no reflow phenomenon. Coronary spasm has been reported following adenosine injection during stress imaging. We report a rare complication with ST segment elevation, following adenosine injection, given for treatment of supraventricular tachycardia. PMID:24581102

  3. Polymerase chain reaction system

    DOEpatents

    Benett, William J.; Richards, James B.; Stratton, Paul L.; Hadley, Dean R.; Milanovich, Fred P.; Belgrader, Phil; Meyer, Peter L.

    2004-03-02

    A portable polymerase chain reaction DNA amplification and detection system includes one or more chamber modules. Each module supports a duplex assay of a biological sample. Each module has two parallel interrogation ports with a linear optical system. The system is capable of being handheld.

  4. Effects of adenosine infusion into renal interstitium on renal hemodynamics

    SciTech Connect

    Pawlowska, D.; Granger, J.P.; Knox, F.G.

    1987-04-01

    This study was designed to investigate the hemodynamic effects of exogenous adenosine in the interstitium of the rat kidney. Adenosine or its analogues were infused into the renal interstitium by means of chronically implanted capsules. In fusion of adenosine decreased glomerular filtration rate (GFR) from 0.81 +/- 0.06 to 0.37 +/- 0.06 ml/min while having no effect on renal blood flow (RBF). The metabolically stable analogue, 2-chloradenosine (2-ClAdo), decreased GFR from 0.73 +/- 0.07 to 021 +/- 0.06 ml/min. Interstitial infusion of theophylline, an adenosine receptor antagonist, completely abolished the effects of adenosine and 2-ClAdo on GFR. The distribution of adenosine, when infused into the renal interstitium, was determined using radiolabeled 5'-(N-ethyl)-carboxamidoadenosine (NECA), a metabolically stable adenosine agonist. After continuous infusion, (/sup 3/H)NECA was distributed throughout the kidney. The effects of NECA to reduce GFR were similar to those of adenosine and 2-ClAdo. They conclude that increased levels of adenosine in the renal interstitium markedly decrease GFR without affecting RBF in steady-state conditions. The marked effects of adenosine agonists during their infusion into the renal interstitium and the complete blockade of these effects by theophylline suggest an extracellular action of adenosine.

  5. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  6. An adenosine nucleoside analogue NITD008 inhibits EV71 proliferation.

    PubMed

    Shang, Luqing; Wang, Yaxin; Qing, Jie; Shu, Bo; Cao, Lin; Lou, Zhiyong; Gong, Peng; Sun, Yuna; Yin, Zheng

    2014-12-01

    Enterovirus 71 (EV71), one of the major causative agents of Hand-Foot-Mouth Disease (HFMD), causes severe pandemics and hundreds of deaths in the Asia-Pacific region annually and is an enormous public health threat. However, effective therapeutic antiviral drugs against EV71 are rare. Nucleoside analogues have been successfully used in the clinic for the treatment of various viral infections. We evaluated a total of 27 nucleoside analogues and discovered that an adenosine nucleoside analogue NITD008, which has been reported to be an antiviral reagent that specifically inhibits flaviviruses, effectively suppressed the propagation of different strains of EV71 in RD, 293T and Vero cells with a relatively high selectivity index. Triphosphorylated NITD008 (ppp-NITD008) functions as a chain terminator to directly inhibit the RNA-dependent RNA polymerase activity of EV71, and it does not affect the EV71 VPg uridylylation process. A significant synergistic anti-EV71 effect of NITD008 with rupintrivir (AG7088) (a protease inhibitor) was documented, supporting the potential combination therapy of NITD008 with other inhibitors for the treatment of EV71 infections.

  7. Adenosine Receptors: Expression, Function and Regulation

    PubMed Central

    Sheth, Sandeep; Brito, Rafael; Mukherjea, Debashree; Rybak, Leonard P.; Ramkumar, Vickram

    2014-01-01

    Adenosine receptors (ARs) comprise a group of G protein-coupled receptors (GPCR) which mediate the physiological actions of adenosine. To date, four AR subtypes have been cloned and identified in different tissues. These receptors have distinct localization, signal transduction pathways and different means of regulation upon exposure to agonists. This review will describe the biochemical characteristics and signaling cascade associated with each receptor and provide insight into how these receptors are regulated in response to agonists. A key property of some of these receptors is their ability to serve as sensors of cellular oxidative stress, which is transmitted by transcription factors, such as nuclear factor (NF)-κB, to regulate the expression of ARs. Recent observations of oligomerization of these receptors into homo- and heterodimers will be discussed. In addition, the importance of these receptors in the regulation of normal and pathological processes such as sleep, the development of cancers and in protection against hearing loss will be examined. PMID:24477263

  8. Adenosine-induced worsening of supraventricular tachycardia

    PubMed Central

    Kunnumpuram, Georgey Koshy; Patel, Ashfaq

    2012-01-01

    An approximately 20-year-old to 30-year-old patient presented with a haemodynamically stable supraventricular tachycardia . The patient was managed with intravenous adenosine primarily, with two bolus doses of 6 and 12 mg. This, however, caused a rare paradoxical surge of tachycardia with mild haemodynamic compromise. The patient further required a combination of Metoprolol and Verapamil administration to slow down and reverse the arrhythmia. Following this the patient remained stable with no further episodes till discharge. PMID:23230260

  9. 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.

  10. Expression of calcitonin gene-related peptide, adenosine A2a receptor and adenosine A1 receptor in experiment rat migraine models

    PubMed Central

    LU, WENXIAN; LI, BIN; CHEN, JINBO; SU, YIPENG; DONG, XIAOMENG; SU, XINYANG; GAO, LIXIANG

    2016-01-01

    A migraine is a disabling neurovascular disorder characterized by a unilateral throbbing headache that lasts from 4 to 72 h. The headache is often accompanied by nausea, vomiting, phonophobia and photophobia, and may be worsened by physical exercise. The trigeminovascular system (TVS) is speculated to have an important role in migraines, although the pathophysiology of this disorder remains to be elucidated. Trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (TNC) are important components of the TVS. Several clinical cases have provided evidence for the involvement of the brainstem in migraine initiation. Electrical stimulation of the trigeminal ganglion (ESTG) in rats can activate TVS during a migraine attack. Calcitonin gene-related peptide (CGRP) is an important vasoactive compound produced following TVS activation. Numerous studies have revealed that adenosine and its receptors have an important role in pain transmission and regulation process. However, only a few studies have examined whether adenosine A2a receptor (A2aR) and adenosine A1 receptor (A1R) are involved in migraine and nociceptive pathways. In the present study, CGRP, A2aR and A1R expression levels were detected in the TG and TNC of ESTG models through reverse transcription-quantitative polymerase chain reaction and western blot analysis. Tianshu capsule (TSC), a type of Chinese medicine, was also used in the ESTG rat models to examine its influence on the three proteins. Results demonstrated that CGRP, A2aR and A1R mediated pain transmission and the regulation process during migraine and the expression of the three proteins was regulated by TSC. PMID:26998280

  11. Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells

    PubMed Central

    Kwak, Choong-Hwan; Lee, Sook-Hyun; Lee, Sung-Kyun; Ha, Sun-Hyung; Suh, Seok-Jong; Kwon, Kyung-Min; Chung, Tae-Wook; Ha, Ki-Tae; Chang, Young-Chae; Lee, Young-Choon; Kim, Dong-Soo; Chang, Hyeun-Wook; Kim, Cheorl-Ho

    2015-01-01

    For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells. PMID:26090845

  12. Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PARP-1 as a cellular target whose interaction is critical for virus biology.

    PubMed

    Liu, Long; Lear, Zoe; Hughes, David J; Wu, Weining; Zhou, En-min; Whitehouse, Adrian; Chen, Hongying; Hiscox, Julian A

    2015-03-23

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry and food security worldwide. The nucleocapsid (N) protein is a major structural protein of PRRSV. The primary function of this protein is to encapsidate the viral RNA genome, and it is also thought to participate in the modulation of host cell biology and recruitment of cellular factors to facilitate virus infection. In order to the better understand these latter roles the cellular interactome of PRRSV N protein was defined using label free quantitative proteomics. This identified several cellular factors that could interact with the N protein including poly [ADP-ribose] polymerase 1 (PARP-1), a cellular protein, which can add adenosine diphosphate ribose to a protein. Use of the PARP-1 small molecule inhibitor, 3-AB, in PRRSV infected cells demonstrated that PARP-1 was required and acted as an enhancer factor for virus biology. Serial growth of PRRSV in different concentrations of 3-AB did not yield viruses that were able to grow with wild type kinetics, suggesting that by targeting a cellular protein crucial for virus biology, resistant phenotypes did not emerge. This study provides further evidence that cellular proteins, which are critical for virus biology, can also be targeted to ablate virus growth and provide a high barrier for the emergence of drug resistance.

  13. Harmine Hydrochloride Triggers G2 Phase Arrest and Apoptosis in MGC-803 Cells and SMMC-7721 Cells by Upregulating p21, Activating Caspase-8/Bid, and Downregulating ERK/Bad Pathway.

    PubMed

    Zhang, Peng; Huang, Chun-Rong; Wang, Wei; Zhang, Xia-Kai; Chen, Jia-Jin; Wang, Juan-Juan; Lin, Chen; Jiang, Jian-Wei

    2016-01-01

    This study aimed to investigate the effects of harmine hydrochloride (HMH) on digestive tumor cells in vitro and its molecular mechanism. MTT assays showed that HMH inhibited the proliferation of some human cancer cell lines and had no obvious inhibitory effects on human LO2 cells. Flow cytometry assays showed that HMH trigged G2 phase arrest in MGC-803 cells and SMMC-7721 cells, while the expression of cyclin A, cyclin B, p21, Myt1, and p-cdc2 (Tyr15) was upregulated. Flow cytometry assays also showed that the percentages of apoptotic cells were increased, the mitochondrial transmembrane potential (ΔΨm) decreased, and the cleavage of caspase-9, caspase-3, and poly (Adenosine diphosphate ribose) polymerase (PARP) were observed, the expression of Bad increased, phospho-Bad (S112) decreased, pro-caspase-8 was cleaved, and Bid (22 kDa) was cleaved. The expression of p-ERK decreased in both cells. In conclusion, these results demonstrated that HMH upregulates the expression of p21, activates Myt1 and inhibits cdc2 by phospho-cdc2 (Y15), and triggers G2 phase arrest in both MGC-803 cells and SMMC-7721 cells. It can also activate the mitochondria-related cell apoptosis pathway through the caspase-8/Bid pathway, inhibiting the ERK/Bad pathway and promoting apoptosis in both of these two cell types.

  14. Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

    PubMed Central

    CHEN, XIAO-MENG; ZHANG, MENG; FAN, PENG-LI; QIN, YU-HUA; ZHAO, HONG-WEI

    2016-01-01

    Previous studies have demonstrated that the benzo[c]phenanthridine alkaloid chelerythrine chloride (CC) has inhibitory effects on various tumors. However, the anticancer activity of CC and its underlying mechanisms have not been elucidated in renal cancer cells. The present study examined the effects of CC on growth inhibition and apoptosis of renal cancer cells in vitro and in vivo. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that CC markedly suppressed the growth of HEK-293 and human renal cancer SW-839 cells in a time- and dose-dependent manner. The xenograft mouse model, which was performed in nude mice, exhibited a reduced tumor growth following CC treatment. In addition, the present study revealed that CC significantly decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, which was accompanied by upregulation of p53, B-cell lymphoma 2 (Bcl-2)-associated X protein, cleaved caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP), and downregulation of Bcl-2, caspase-3 and PARP. Furthermore, the use of PD98059, a specific mitogen-activated protein kinase kinase inhibitor, potentiated the proapoptotic effects of CC, which indicated that CC may induce apoptosis in renal cancer cells partly via inhibition of ERK activity. Overall, the results of the present study demonstrated that CC may be developed as a potential anticancer treatment for patients with renal cancer. PMID:27313717

  15. Heat Inactivation of Garlic (Allium sativum) Extract Abrogates Growth Inhibition of HeLa Cells.

    PubMed

    Chintapalli, Renuka; Murray, Matthew J J; Murray, James T

    2016-07-01

    The potential anticancer properties of garlic (Allium sativum) may depend on the method of preparation and its storage. Storage of garlic has not been thoroughly investigated to determine whether anticancer properties are retained. Garlic was prepared and processed to mimic normal options for storage and preparation for consumption. Cytotoxicity was determined by crystal violet assay and mechanisms of cytotoxicity were established by microscopy, SDS-PAGE, and Western immunoblotting. Significant (P < 0.0001) cytotoxicity was observed in all preparations, except with boiled (cooked) garlic. Depending on the method of storage, garlic extract induced either type I or type II programmed cell death, detectable by caspase 9 cleavage, or Poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage and LC3-II accumulation, respectively. The conflicting literature on the anticancer properties of garlic may be explained by differences in processing and storage. This study has highlighted that the potency of the antiproliferative properties of cooked garlic, compared to the uncooked form, is diminished in HeLa cells. PMID:27176674

  16. Role of Biomarkers in the Development of PARP Inhibitors.

    PubMed

    Ganguly, Bratati; Dolfi, Sonia C; Rodriguez-Rodriguez, Lorna; Ganesan, Shridar; Hirshfield, Kim M

    2016-01-01

    Defects in DNA repair lead to genomic instability and play a critical role in cancer development. Understanding the process by which DNA damage repair is altered or bypassed in cancer may identify novel therapeutic targets and lead to improved patient outcomes. Poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) has an important role in DNA repair, and novel therapeutics targeting PARP1 have been developed to treat cancers with defective DNA repair pathways. Despite treatment successes with PARP inhibitors (PARPi), intrinsic and acquired resistances have been observed. Preclinical studies and clinical trials in cancer suggest that combination therapy using PARPi and platinating agents is more effective than monotherapy in circumventing drug resistance mechanisms. Additionally, identification of biomarkers in response to PARPi will lead to improved patient selection for targeted cancer treatment. Recent technological advances have provided the necessary tools to examine many potential avenues to develop such biomarkers. This review examines the mechanistic rationale of PARP inhibition and potential biomarkers in their development for personalized therapy.

  17. Role of Biomarkers in the Development of PARP Inhibitors

    PubMed Central

    Ganguly, Bratati; Dolfi, Sonia C.; Rodriguez-Rodriguez, Lorna; Ganesan, Shridar; Hirshfield, Kim M.

    2016-01-01

    Defects in DNA repair lead to genomic instability and play a critical role in cancer development. Understanding the process by which DNA damage repair is altered or bypassed in cancer may identify novel therapeutic targets and lead to improved patient outcomes. Poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) has an important role in DNA repair, and novel therapeutics targeting PARP1 have been developed to treat cancers with defective DNA repair pathways. Despite treatment successes with PARP inhibitors (PARPi), intrinsic and acquired resistances have been observed. Preclinical studies and clinical trials in cancer suggest that combination therapy using PARPi and platinating agents is more effective than monotherapy in circumventing drug resistance mechanisms. Additionally, identification of biomarkers in response to PARPi will lead to improved patient selection for targeted cancer treatment. Recent technological advances have provided the necessary tools to examine many potential avenues to develop such biomarkers. This review examines the mechanistic rationale of PARP inhibition and potential biomarkers in their development for personalized therapy. PMID:26997874

  18. Effect of adenosine and adenosine analogs on ( sup 14 C)aminopyrine accumulation by rabbit parietal cells

    SciTech Connect

    Ota, S.; Hiraishi, H.; Terano, A.; Mutoh, H.; Kurachi, Y.; Shimada, T.; Ivey, K.J.; Sugimoto, T. )

    1989-12-01

    Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on (14C)aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. (14C)Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated (14C)aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on (14C)aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased (14C)aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.

  19. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 2. Expression of receptor mRNA in resting and lipopolysaccharide-activated mouse RAW 264.7 macrophages.

    PubMed

    Streitová, D; Hofer, M; Holá, J; Vacek, A; Pospísil, M

    2010-01-01

    Expression of mRNA for adenosine receptor subtypes A(1), A(2a), A(2b), and A(3) in normal and lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages has been investigated using the method of quantitative real-time polymerase chain reaction. The results have shown a very low, unquantifiable expression of adenosine A(1) receptor mRNA in both normal and LPS-activated macrophages. The other three adenosine receptor mRNAs have been found to be expressed at various but always quantifiable levels. Activation of the macrophages by LPS induced upregulation of the expression of adenosine receptor A(2a) and A(2b) mRNA, whereas the expression of adenosine receptor A(3) mRNA was downregulated. Unstimulated macrophages exhibited a high expression of the A(2b) adenosine receptor mRNA. The findings are discussed from the point of view of the antiinflammatory and hematopoiesis-stimulating roles of the adenosine receptor signaling.

  20. 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

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

    SciTech Connect

    Zoghbi, W.A. )

    1991-07-01

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

  2. Measurement of plasma adenosine concentration: methodological and physiological considerations

    SciTech Connect

    Gewirtz, H.; Brown, P.; Most, A.S.

    1987-05-01

    This study tested the hypothesis that measurements of plasma adenosine concentration made on samples of blood obtained in dipyridamole and EHNA (i.e., stopping solution) may be falsely elevated as a result of ongoing in vitro production and accumulation of adenosine during sample processing. Studies were performed with samples of anticoagulated blood obtained from anesthesized domestic swine. Adenosine concentration of ultra filtrated plasma was determined by HPLC. The following parameters were evaluated: (i) rate of clearance of (/sup 3/H)adenosine added to plasma, (ii) endogenous adenosine concentration of matched blood samples obtained in stopping solution alone, stopping solution plus EDTA, and perchloric acid (PCA), (iii) plasma and erythrocyte endogenous adenosine concentration in nonhemolyzed samples, and (iv) plasma adenosine concentration of samples hemolyzed in the presence of stopping solution alone or stopping solution plus EDTA. We observed that (i) greater than or equal to 95% of (/sup 3/H)adenosine added to plasma is removed from it by formed elements of the blood in less than 20 s, (ii) plasma adenosine concentration of samples obtained in stopping solution alone is generally 10-fold greater than that of matched samples obtained in stopping solution plus EDTA, (iii) deliberate mechanical hemolysis of blood samples obtained in stopping solution alone resulted in substantial augmentation of plasma adenosine levels in comparison with matched nonhemolyzed specimens--addition of EDTA to stopping solution prevented this, and (iv) adenosine content of blood samples obtained in PCA agreed closely with the sum of plasma and erythrocyte adenosine content of samples obtained in stopping solution plus EDTA.

  3. Role of adenosine receptor subtypes in methamphetamine reward and reinforcement.

    PubMed

    Kavanagh, Kevin A; Schreiner, Drew C; Levis, Sophia C; O'Neill, Casey E; Bachtell, Ryan K

    2015-02-01

    The neurobiology of methamphetamine (MA) remains largely unknown despite its high abuse liability. The present series of studies explored the role of adenosine receptors on MA reward and reinforcement and identified alterations in the expression of adenosine receptors in dopamine terminal areas following MA administration in rats. We tested whether stimulating adenosine A1 or A2A receptor subtypes would influence MA-induced place preference or MA self-administration on fixed and progressive ratio schedules in male Sprague-Dawley rats. Stimulation of either adenosine A1 or A2A receptors significantly reduced the development of MA-induced place preference. Stimulating adenosine A1, but not A2A, receptors reduced MA self-administration responding. We next tested whether repeated experimenter-delivered MA administration would alter the expression of adenosine receptors in the striatal areas using immunoblotting. We observed no change in the expression of adenosine receptors. Lastly, rats were trained to self-administer MA or saline for 14 days and we detected changes in adenosine A1 and A2A receptor expression using immunoblotting. MA self-administration significantly increased adenosine A1 in the nucleus accumbens shell, caudate-putamen and prefrontal cortex. MA self-administration significantly decreased adenosine A2A receptor expression in the nucleus accumbens shell, but increased A2A receptor expression in the amygdala. These findings demonstrate that MA self-administration produces selective alterations in adenosine receptor expression in the nucleus accumbens shell and that stimulation of adenosine receptors reduces several behavioral indices of MA addiction. Together, these studies shed light onto the neurobiological alterations incurred through chronic MA use that may aid in the development of treatments for MA addiction.

  4. Caffeine intensifies taste of certain sweeteners: role of adenosine receptor.

    PubMed

    Schiffman, S S; Diaz, C; Beeker, T G

    1986-03-01

    Caffeine, a potent antagonist of adenosine receptors, potentiates the taste of some but not all sweeteners. It significantly enhances the taste of acesulfam-K, neohesperidin dihydrochalcone, d-tryptophan, thaumatin, stevioside, and sodium saccharin. Adenosine reverses the enhancement. Caffeine has no effect on aspartame, sucrose, fructose, and calcium cyclamate. These results suggest that the inhibitory A1 adenosine receptor plays an important local role in modulating the taste intensity of certain sweeteners and that several transduction mechanisms mediate sweet taste.

  5. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment.

    PubMed

    Ohta, Akio

    2016-01-01

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

  6. Adenosine analogs inhibit fighting in isolated male mice

    SciTech Connect

    Palmour, R.M.; Lipowski, C.J.; Simon, C.K.; Ervin, F.R.

    1989-01-01

    The potent adenosine analogs N-ethylcarboxamide adenosine (NECA) and phenylisopropyladenosine (PIA) inhibit fighting and associated agonistic behaviors in isolated male mice. These effects are reversed by methylxanthines; moderate doses of NECA which inhibit fighting have minimal effects on spontaneous locomotor activity. At very low doses, both NECA and PIA increase fighting in parallel with previously reported increases of motor activity. Brain levels of (/sup 3/H)-NECA and (/sup 3/H)-PIA achieved at behaviorally effective doses suggest an involvement of adenosine receptors. The biochemical mechanism of adenosine receptor action with respect to fighting is unknown, but may include neuromodulatory effects on the release of other, more classical neurotransmitters.

  7. Adenosine signaling: good or bad in erectile function?

    PubMed

    Wen, Jiaming; Xia, Yang

    2012-04-01

    The erectile status of penile tissue is governed largely by the tone of cavernosal smooth muscle cells, which is determined by the balance of vascular relaxants and constrictors. Vascular relaxants play a key role in regulating the tone of cavernosal smooth muscle and thus the initiation and maintenance of penile erection. Early studies drew attention to the potential role of adenosine signaling in this process. However, the serendipitous discovery of the effect of sildenafil on erectile physiology drew more attention toward nitric oxide (NO) as a vasodilator in the process of penile erection, and a recently discovered, unexpected erectile phenotype of adenosine deaminase-deficient mice reemphasizes the importance of adenosine as a key regulatory of erectile status. Adenosine, like NO, is a potent and short-lived vasorelaxant that functions via cyclic nucleotide second messenger signaling to promote smooth muscle relaxation. Recent studies reviewed here show that adenosine functions to relax the corpus cavernosum and promote penile erection. Excess adenosine in penile tissue contributes to the disorder called priapism, and impaired adenosine signaling is associated with erectile dysfunction. More recent research summarized in this review reveals that adenosine functions as a key endogenous vasodilator in the initiation and maintenance of normal penile erection. This new insight highlights adenosine signaling pathways operating in penile tissue as significant therapeutic targets for the treatment of erectile disorders.

  8. 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.... Simultaneous measurements of platelet aggregation and ATP release are used to evaluate platelet...

  9. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGES

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Errormore » rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  10. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

    PubMed

    McInerney, Peter; Adams, Paul; Hadi, Masood Z

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. PMID:25197572

  11. Introduction to Adenosine Receptors as Therapeutic Targets

    PubMed Central

    Jacobson, Kenneth A.

    2012-01-01

    Adenosine acts as a cytoprotective modulator in response to stress to an organ or tissue. Although short-lived in the circulation, it can activate four sub-types of G protein-coupled adenosine receptors (ARs): A1, A2A, A2B, and A3. The alkylxanthines caffeine and theophylline are the prototypical antagonists of ARs, and their stimulant actions occur primarily through this mechanism. For each of the four AR subtypes, selective agonists and antagonists have been introduced and used to develop new therapeutic drug concepts. ARs are notable among the GPCR family in the number and variety of agonist therapeutic candidates that have been proposed. The selective and potent synthetic AR agonists, which are typically much longer lasting in the body than adenosine, have potential therapeutic applications based on their anti-inflammatory (A2A and A3), cardioprotective (preconditioning by A1 and A3 and postconditioning by A2B), cerebroprotective (A1 and A3), and antinociceptive (A1) properties. Potent and selective AR antagonists display therapeutic potential as kidney protective (A1), antifibrotic (A2A), neuroprotective (A2A), and antiglaucoma (A3) agents. AR agonists for cardiac imaging and positron-emitting AR antagonists are in development for diagnostic applications. Allosteric modulators of A1 and A3 ARs have been described. In addition to the use of selective agonists/antagonists as pharmacological tools, mouse strains in which an AR has been genetically deleted have aided in developing novel drug concepts based on the modulation of ARs. PMID:19639277

  12. Adenosine signaling in normal and sickle erythrocytes and beyond

    PubMed Central

    Zhang, Yujin; Xia, Yang

    2012-01-01

    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 A2B receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O2 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 A2A 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 progression

  13. Characterization of adenosine binding proteins in human placental membranes

    SciTech Connect

    Hutchison, K.A.

    1989-01-01

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with ({sup 3}H) -N-ethylcarboxamidoadenosine (({sup 3}H)NECA). This site is similar to the adenosine A{sub 2} receptor. We call this site the adenosine A{sub 2}-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A{sub 1} receptor. The soluble adenosine A{sub 2}-like binding site cannot be detected without a rapid assay. Binding to the adenosine A{sub 1} receptor with ({sup 3}H)-2-chloroadenosine and ({sup 3}H)NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A{sub 1} specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5{prime}-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands ({sup 3}H)-cyclohexyladenosine ({sup 3}H) cylopentylxanthine revealed adenosine A{sub 1} agonist and antagonist potency orders. We have purified the adenosine A{sub 2}-like binding site. The adenosine A{sub 2}-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 {Angstrom}. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A{sub 2}-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine.

  14. Polymerase chain reaction

    SciTech Connect

    Arnhelm, N. ); Levenson, C.H. )

    1990-10-01

    This paper discusses the polymerase chain reaction (PCR) an in-vitro method of amplifying DNA sequences. Beginning with DNA of any origin- bacterial, viral, plant, or animal- PCR can increase the amount of a DNA sequence hundreds of millions to billions of times. The procedure can amplify a targeted sequence even when it makes up less than one part in a million of the total initial sample. PCR is an enzymatic process that is carried out in discrete cycles of amplification, each of which can double the amount of target DNA in the sample. Thus, n cycles can produce 2{sup n} times as much target as was present to begin with. This paper discusses how PCR has had an impact on molecular biology, human genetics, infectious and genetic disease diagnosis, forensic science, and evolutionary biology.

  15. Norepinephrines effect on adenosine transport in the proximal straight tubule

    SciTech Connect

    Barfuss, D.W.; McCann, W.P.; Katholi, R.E.

    1986-03-01

    The effect of norepinephrine on C/sup 14/-adenosine transport in the rabbit proximal tubule (S/sub 2/) was studied. The transepithelial transport of adenosine (0.02 mM0 from lumin to bathing solution was measured by its rate of appearance (J/sub A/) in the bathing solution and by its disappearances (J/sub D/) from the luminal fluid. Norepinephrine (0.24 ..mu..M) was added to the bathing solution after a control flux period. After three samples from the experiment period the tubules were quickly harvested and the cellular concentration of C/sup 14/-adenosine was determined. The high cellular adenosine concentration and th marked difference in adenosine appearance rate in the bathing solution compared to the luminal disappearance rate indicates the absorbed adenosine is trapped in the cells. This trapping may be due to adenosine metabolism or difficulty of crossing the basolateral membrane. Whichever is the case, norepinephrine appears to stimulate movement of adenosine or its metabolites into the bathing solution across the basolateral membrane.

  16. Comorbidities in Neurology: Is Adenosine the Common Link?

    PubMed Central

    Boison, Detlev; Aronica, Eleonora

    2015-01-01

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

  17. Effect of theophylline on adenosine production in the canine myocardium

    SciTech Connect

    McKenzie, J.E.; Steffen, R.P.; Haddy, F.J.

    1987-01-01

    Adenosine is thought to participate in local regulation of coronary blood flow. However, competitive antagonists of adenosine fail to block myocardial active hyperemia. The authors examined the effect of locally administered theophylline on active hyperemia and myocardial adenosine production during intracoronary isoproterenol infusion in the dog heart. Isoproterenol decreased coronary resistance and increased myocardial adenosine production. Infusion of theophylline at a rate that attenuated the vasodilator response to exogenously administered adenosine failed to attenuate the increase in coronary blood flow produced by isoproterenol. However, theophylline plus isoproterenol production greater increases in myocardial adensine production than isoproterenol alone. The curves relating resistance and adenosine in the presence of theophylline fell to the right of those in the absence of theophylline. These findings suggest that the failure of theophylline to attenuate isoproterenol hyperemia in the dog heart results at least in part from an increase in adenosine concentration at the arteriole to a level beyond that blocked by this competitive antagonist and that adenosine may in fact play a role in isoproterenol-induced active hyperemia.

  18. p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis.

    PubMed

    Khanal, Prem; Oh, Won-Keun; Yun, Hyo Jeong; Namgoong, Gwang Mo; Ahn, Sang-Gun; Kwon, Seong-Min; Choi, Hoo-Kyun; Choi, Hong Seok

    2011-04-01

    Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells. PMID:21216846

  19. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

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

    2011-12-31

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

  20. Serum adenosine deaminase activity in cutaneous anthrax

    PubMed Central

    Sunnetcioglu, Mahmut; Karadas, Sevdegul; Aslan, Mehmet; Ceylan, Mehmet Resat; Demir, Halit; Oncu, Mehmet Resit; Karahocagil, Mustafa Kasım; Sunnetcioglu, Aysel; Aypak, Cenk

    2014-01-01

    Background Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. Material/Methods Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. Results Serum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (p<0.001). A positive correlation was observed between ADA activity and lymphocyte counts (r=0.589, p=0.021) in the patient group. Conclusions This study suggests that serum ADA could be used as a biochemical marker in cutaneous anthrax. PMID:24997584

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

    PubMed

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

    2016-07-01

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

  2. 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

  3. Identification of possible adenosine receptors in vascular smooth muscle

    SciTech Connect

    Doctrow, S.R.

    1985-01-01

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

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

    SciTech Connect

    Tritsch, G.L.

    1985-01-01

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

  5. 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

  6. Adenosine receptor ligands: differences with acute versus chronic treatment

    PubMed Central

    Jacobson, Kenneth A.; von Lubitz, Dag K. J. E.; Daly, John W.; Fredholm, Bertil B.

    2012-01-01

    Adenosine receptors have been the target of intense research with respect to potential use of selective ligands in a variety of therapeutic areas. Caffeine and theophylline are adenosine receptor antagonists, and over the past three decades a wide range of selective agonists and antagonists for adenosine receptor subtypes have been developed. A complication to the therapeutic use of adenosine receptor ligands is the observation that the effects of acute administration of a particular ligand can be diametrically opposite to the chronic effects of the same ligand. This ‘effect inversion’ is discussed here by Ken Jecobson and colleagues, and has been observed for effects on cognitive processes, seizures and ischaemic damage. PMID:8936347

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

    PubMed Central

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

    2015-01-01

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

  8. d-Propranolol prevents adenosine formation associated with myocardial hypoperfusion.

    PubMed

    Wangler, R D; Peterson, W P; Sparks, H V

    1989-03-01

    d-Propranolol eliminates the increased adenine nucleoside release from hypoperfused hearts [R. D. Wangler, D. F. DeWitt, and H. V. Sparks, Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H330-H336, 1984]. To determine whether d-propranolol reduces adenosine formation or adenosine release into the vascular compartment, we measured myocardial tissue adenosine (TADO). Decreased formation would lower TADO, whereas decreased release would elevate TADO. Reduction of perfusion pressure by 50% reduced coronary flow (CF), venous oxygen tension (PVO2), and myocardial oxygen consumption (MVO2) by approximately 40, 25, and 35%, respectively. Total adenosine and inosine released during 30 min of hypoperfusion increased 10- and 5-fold, respectively. Also, TADO increased from 2.68 +/- 0.37 to 5.17 +/- 0.67 nmol/g (P less than 0.05). In the presence of d-propranolol, the same reduction in perfusion pressure caused a similar decrease in CF and MVO2. d-Propranolol eliminated the release of adenosine and inosine associated with hypoperfusion. TADO after 30 min of hypoperfusion plus d-propranolol was not significantly increased (3.27 +/- 0.40 nmol/g) and was significantly less than hypoperfused hearts. When severe hypoperfusion was created by reducing perfusion pressure 75%, adenosine release still did not increase if d-propranolol was present. When adenosine release was plotted as a function of oxygen supply-consumption, they were related in a hyperbolic fashion. Despite the severity of hypoperfusion, in the presence of d-propranolol the supply-to-consumption ratio was similar to that of the control perfusion group (no drug). We conclude that d-propranolol blocks nucleoside formation during hypoperfusion by reducing oxygen demand such that a reduction of oxygen supply no longer stimulates adenosine formation. PMID:2923237

  9. A label-free fluorescent molecular beacon based on DNA-templated silver nanoclusters for detection of adenosine and adenosine deaminase.

    PubMed

    Zhang, Min; Guo, Su-Miao; Li, Ying-Ru; Zuo, Peng; Ye, Bang-Ce

    2012-06-01

    A simple and reliable fluorescent molecular beacon is developed utilizing DNA-templated silver nanoclusters as a signal indicator and adenosine triphosphate (ATP) and adenosine deaminase as mechanical activators.

  10. A selective adenosine sensor derived from a triplex DNA aptamer.

    PubMed

    Patel, Mayurbhai; Dutta, Avishek; Huang, Haidong

    2011-07-01

    The aim of this study is to develop a selective adenosine aptamer sensor using a rational approach. Unlike traditional RNA aptamers developed from SELEX, duplex DNA containing an abasic site can function as a general scaffold to rationally design aptamers for small aromatic molecules. We discovered that abasic site-containing triplex DNA can also function as an aptamer and provide better affinity than duplex DNA aptamers. A novel adenosine aptamer sensor was designed using such a triplex. The aptamer is modified with furano-dU in the binding site to sense the binding. The sensor bound adenosine has a dissociation constant of 400 nM, more than tenfold stronger than the adenosine aptamer developed from SELEX. The binding quenched furano-dU fluorescence by 40%. It was also demonstrated in this study that this sensor is selective for adenosine over uridine, cytidine, guanosine, ATP, and AMP. The detection limit of this sensor is about 50 nM. The sensor can be used to quantify adenosine concentrations between 50 nM and 2 μM. PMID:21547431

  11. Intrarenal blood flow distribution during adenosine-mediated vasoconstriction.

    PubMed

    Macias, J F; Fiksen-Olsen, M; Romero, J C; Knox, F G

    1983-01-01

    Intrarenal infusion of adenosine induces an initial vasoconstriction followed by a subsequent vasodilation. The intrarenal distribution of blood flow in the vasoconstriction phase is unknown. The present study was undertaken to assess the effect of intrarenal infusion of adenosine on intracortical distribution of renal blood flow during both the vasoconstriction and vasodilation phases. Renal blood flow distribution was measured with radiolabeled microspheres in anesthetized sodium-depleted dogs before and during the early vasoconstriction phase and the late vasodilation phase of intrarenal infusion of adenosine. During the vasoconstriction phase, there was a uniform decrease in blood flow in each renal cortical zone. In the late phase of adenosine infusion, there was a significant increase in deep cortical flow without significant changes in superficial cortical flow compared with control. The effects of adenosine were also compared with those exerted by norepinephrine in which decreased blood flow was demonstrated in all zones. We conclude that the vasoconstrictor phase of adenosine infusion is characterized by a uniform reduction of renal blood flow to all cortical zones, whereas the vasodilator phase is characterized by a selective deep cortical vasodilation.

  12. 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

  13. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

    PubMed Central

    Guranowski, A; Günther Sillero, M A; Sillero, A

    1994-01-01

    Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605

  14. Adaptations in adenosine signaling in drug dependence: therapeutic implications.

    PubMed

    Hack, Stephen P; Christie, Macdonald J

    2003-01-01

    Adenosine is an important endogenous purine neuromodulator in the central nervous system that modulates many important cellular processes in neurons. The physiological effects of adenosine are transduced through four pharmacologically classified receptor types i.e., A1, A2A, A2B and A3. All adenosine receptors are G-protein coupled receptors (GPCR) of the type 1 variety. Adaptations in adenosine signaling have been implicated in a wide range of pathophysiological processes, such as epilepsies, sleep disorders, pain, and drug addictions. Knowledge relating to the etiology of addictive processes is far from complete, and as a result the therapeutic options to deal with drug dependence issues are limited. Drugs of abuse mediate their effects through many distinct cellular effectors, such as neurotransmitter transporters, ion channels, and receptor proteins. However, a unifying feature of the major drugs of abuse-i.e., opiates, cocaine, and alcohol-is that they all directly or indirectly modulate adenosine signaling in neurons. Agents targeting adenosine receptors may therefore offer novel avenues for the development of therapies to manage or treat addictions. A consistent cellular adaptation to long-term drug use is the up- or down-regulation of signaling pathways driven by adenylyl cyclase/cyclic AMP (cAMP) in several brain regions linked to addiction. Withdrawal from mu-opioids or cocaine following their chronic administration leads to an upregulation of adenylyl cyclase-mediated signaling, resulting in high levels of cAMP. Cyclic AMP produced in this way acts as a substrate for the endogenous production of adenosine. Increased levels of endogenous adenosine interact with presynaptic A1 receptors to inhibit the excessive neuronal excitation often seen during morphine/cocaine withdrawal. These pre-clinical findings fit well with other data indicating that drugs which boost endogenous adenosine levels or directly interact with inhibitory A1 receptors can alleviate

  15. Interstitial adenosine concentration is increased by dipyridamole

    SciTech Connect

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

    1986-03-01

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

  16. Protective effects of sirtuins in cardiovascular diseases: from bench to bedside

    PubMed Central

    Winnik, Stephan; Auwerx, Johan; Sinclair, David A.; Matter, Christian M.

    2015-01-01

    Sirtuins (Sirt1–Sirt7) comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent enzymes. While deacetylation reflects their main task, some of them have deacylase, adenosine diphosphate-ribosylase, demalonylase, glutarylase, and desuccinylase properties. Activated upon caloric restriction and exercise, they control critical cellular processes in the nucleus, cytoplasm, and mitochondria to maintain metabolic homeostasis, reduce cellular damage and dampen inflammation—all of which serve to protect against a variety of age-related diseases, including cardiovascular pathologies. This review focuses on the cardiovascular effects of Sirt1, Sirt3, Sirt6, and Sirt7. Most is known about Sirt1. This deacetylase protects from endothelial dysfunction, atherothrombosis, diet-induced obesity, type 2 diabetes, liver steatosis, and myocardial infarction. Sirt3 provides beneficial effects in the context of left ventricular hypertrophy, cardiomyopathy, oxidative stress, metabolic homeostasis, and dyslipidaemia. Sirt6 is implicated in ameliorating dyslipidaemia, cellular senescence, and left ventricular hypertrophy. Sirt7 plays a role in lipid metabolism and cardiomyopathies. Most of these data were derived from experimental findings in genetically modified mice, where NFκB, Pcsk9, low-density lipoprotein-receptor, PPARγ, superoxide dismutase 2, poly[adenosine diphosphate-ribose] polymerase 1, and endothelial nitric oxide synthase were identified among others as crucial molecular targets and/or partners of sirtuins. Of note, there is translational evidence for a role of sirtuins in patients with endothelial dysfunction, type 1 or type 2 diabetes and longevity. Given the availability of specific Sirt1 activators or pan-sirtuin activators that boost levels of the sirtuin cofactor NAD+, we anticipate that this field will move quickly from bench to bedside. PMID:26112889

  17. Nicotinamide augments the survival and incidence of apoptosis in glioma cells following photodynamic therapy in vitro

    NASA Astrophysics Data System (ADS)

    Bisland, Stuart K.; Modi, Nayan; Wilson, Brian C.

    2004-10-01

    The ability to customize photodynamic therapy (PDT) parameters with regards to timing and dosing of administered drug and light can be beneficial in determining target specificity and mode of cell death. Sustained, low level PDT or metronomic PDT (mPDT) may afford enhanced apoptotic cell death. This is of particular importance when considering PDT for the treatment of brain tumors as unlike apoptosis, necrotic cell death often leads to inflammation with increased intracranial pressure. The ability, therefore, to 'fine tune' PDT in favour of apoptosis is paramount. We have studied both acute (one time treatment) PDT (aPDT) and mPDT delivery strategies in combination with nicotinamide (NA) in an attempt to maximize the number of tumor cells dieing by apoptosis. Using several different glioma cell lines (9L, U87-MG and CNS-1) we now confirm that NA provides a dose-dependent (0.1-0.5 mM) increase in apoptotic cells following d-aminolevulinic acid-mediated aPDT or mPDT. Furthermore, using the 9L cell line stably transfected with the luciferase gene, NA was shown to delay the depletion of bioluminscence signal in aPDT and mPDT treated cells, inferring that adenosine triphosphate levels are maintained for longer following NA treatment. NA has previously been reported as promoting neuronal and vascular cell survival in normal brain following a number of neurological insults in which reactive oxygen species are implicated including, stroke, Alzheimer's disease and toxin-induced lesions. It is likely that the effects of NA reflect its capacity as an antioxidant as well as its ability to inhibit poly (adenosine diphosphate-ribose) polymerase-mediated depletion of ATP. Our results indicate that NA may prove therapeutically advantageous when used in combination with PDT treatment of brain tumors.

  18. Production of RNA by a polymerase protein encapsulated within phospholipid vesicles

    NASA Technical Reports Server (NTRS)

    Chakrabarti, A. C.; Breaker, R. R.; Joyce, G. F.; Deamer, D. W.

    1994-01-01

    Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.

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

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

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

  20. Role of A3 adenosine receptor in diabetic neuropathy.

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2010-01-01

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

  2. [Vascular effects of adenosine-triphosphate].

    PubMed

    Colson, P; Saussine, M; Gaba, S; Sequin, J; Chaptal, P A; Roquefeuil, B

    1991-01-01

    This study assessed the effects of adenosine triphosphate (ATP) on systemic vascular resistances during the hypothermic cardiopulmonary bypass phase of cardiac surgery. Twenty patients scheduled for cardiac surgery were randomly divided into an ATP group (n = 10), and a placebo group (n = 10). Anaesthesia was similar for all the patients (diazepam, fentanyl and pancuronium). During the heart arrest phase, and as soon as the arterial pressure, the level in the venous return reservoir, and the pump flow rate had all been in steady state for 5 min, ATP or placebo was injected into the venous line of the oxygenator. Injection speed was doubled every three minutes, twice. The following ATP doses were administered: 0.012, 0.025 and 0.05 mg.kg-1.min-1. The level in the venous return reservoir was kept constant. Mean arterial pressure (MAP) and pump flow rate (DP) were assessed every half minute. Systemic vascular resistances were calculated with the relationship MAP/DP. Changes in vascular capacitance were directly proportional to changes in DP as the heart had been excluded, and all the blood returned to the pump, the blood volume being kept constant. MAP and DP remained unchanged in the placebo group. In the opposite ATP induced a dose-related systemic vasodilation: MAP decreased from 82.8 +/- 12.5 mmHg (control) to 66.0 +/- 14.8 mmHg, 59.8 +/- 10.6 mmHg, and 49.0 +/- 4.7 mmHg with 0.012, 0.025 and 0.05 mg.kg-1.min-1 ATP respectively. The MAP returned to preinfusion control levels when the ATP infusion was discontinued (90.0 +/- 17.8 mmHg). The DP, and therefore venous return, did not change, neither during ATP infusion, nor after its discontinuation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1854051

  3. 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.

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2002-05-10

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

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

    PubMed

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

    2002-05-10

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

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

    PubMed

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

    1985-11-01

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

  9. Adenosine signaling and the regulation of chronic lung disease

    PubMed Central

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

    2009-01-01

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

  10. Effect of adenosine and inosine on ureagenesis in hepatocytes.

    PubMed Central

    Guinzberg, R; Laguna, I; Zentella, A; Guzman, R; Piña, E

    1987-01-01

    Adenosine and inosine produced a dose-dependent stimulation of ureagenesis in isolated rat hepatocytes. Hypoxanthine, xanthine and uric acid were without effect. Half-maximally effective concentrations were 0.08 microM for adenosine and 5 microM for inosine. Activation of ureagenesis by both nucleosides had the following characteristics: (a) it was observed with either glutamine or (NH4)2CO3, provided that glucose was present; (b) it was not detected when glucose was replaced by lactate plus oleate; (c) it was mutually antagonized by glucagon, but not by adrenaline; and (d) it was dependent on Ca2+. We suggest that the action of adenosine and inosine on ureagenesis might be of physiological significance. PMID:3663162

  11. Adenosine receptor agonists for promotion of dermal wound healing

    PubMed Central

    Valls, María D.; Cronstein, Bruce N.; Montesinos, M. Carmen

    2009-01-01

    Wound healing is a dynamic and complex process that involves a well coordinated, highly regulated series of events including inflammation, tissue formation, revascularization and tissue remodeling. However, this orderly sequence is impaired in certain pathophysiological conditions such as diabetes mellitus, venous insufficiency, chronic glucocorticoid use, aging and malnutrition. Together with proper wound care, promotion of the healing process is the primary objective in the management of chronic poorly healing wounds. Recent studies have demonstrated that A2A adenosine receptor agonists promote wound healing in normal and diabetic animals and one such agonist, Sonedenoson, is currently being evaluated as a prospective new therapy of diabetic foot ulcers. We will review the mechanisms by which adenosine receptor activation affects the function of the cells and tissues that participate in wound healing, emphasizing the potential beneficial impact of adenosine receptor agonists in diabetic impaired healing. PMID:19041853

  12. Norovirus Proteinase-Polymerase and Polymerase Are Both Active Forms of RNA-Dependent RNA Polymerase

    PubMed Central

    Belliot, Gaël; Sosnovtsev, Stanislav V.; Chang, Kyeong-Ok; Babu, Vijay; Uche, Uzo; Arnold, Jamie J.; Cameron, Craig E.; Green, Kim Y.

    2005-01-01

    In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro−Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro−Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis. PMID:15681440

  13. Role of adenosine as adjunctive therapy in acute myocardial infarction.

    PubMed

    Forman, Mervyn B; Stone, Gregg W; Jackson, Edwin K

    2006-01-01

    Although early reperfusion and maintained patency is the mainstay therapy for ST elevation myocardial infarction, experimental studies demonstrate that reperfusion per se induces deleterious effects on viable ischemic cells. Thus "myocardial reperfusion injury" may compromise the full potential of reperfusion therapy and may account for unfavorable outcomes in high-risk patients. Although the mechanisms of reperfusion injury are complex and multifactorial, neutrophil-mediated microvascular injury resulting in a progressive decrease in blood flow ("no-reflow" phenomenon) likely plays an important role. Adenosine is an endogenous nucleoside found in large quantities in myocardial and endothelial cells. It activates four well-characterized receptors producing various physiological effects that attenuate many of the proposed mechanisms of reperfusion injury. The cardio-protective effects of adenosine are supported by its role as a mediator of pre- and post-conditioning. In experimental models, administration of adenosine in the peri-reperfusion period results in a marked reduction in infarct size and improvement in ventricular function. The cardioprotective effects in the canine model have a narrow time window with the drug losing its effect following three hours of ischemia. Several small clinical studies have demonstrated that administration of adenosine with reperfusion therapy reduces infarct size and improves ventricular function. In the larger AMISTAD and AMISTAD II trials a 3-h infusion of adenosine as an adjunct to reperfusion resulted in a striking reduction in infarct size (55-65%). Post hoc analysis of AMISTAD II showed that this was associated with significantly improved early and late mortality in patients treated within 3.17 h of symptoms. An intravenous infusion of adenosine for 3 h should be considered as adjunctive therapy in high risk-patients undergoing reperfusion therapy. PMID:16961725

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  15. 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

  16. Phosphorylation of adenosine with trimetaphosphate under simulated prebiotic conditions.

    PubMed

    Cheng, Changmei; Fan, Chang; Wan, Rong; Tong, Chunyuan; Miao, Zhiwei; Chen, Jing; Zhao, Yufen

    2002-06-01

    The phosphorylation of adenosine with trimetaphosphate in solution, in solid phase and using wet-dry cycles was carried out and it was found that wet-dry cycles were the most efficient. The catalytic effects of some metal ions on the phosphorylation were also studied and it was discovered that Ni(II) is the most effective. The combination of wet-dry cycles (4 cycles) and catalysis by Ni(II) led to an unprecedented high conversion of adenosine to phosphorylated products (30%) near neutral pH. The main phosphorylated products were 2',3'-cyclic AMP (10.4%) and 5'-ATP (13.0%). PMID:12227426

  17. S-Adenosylhomocysteine toxicity in normal and adenosine kinase-deficient lymphoblasts of human origin

    PubMed Central

    Kredich, Nicholas M.; Hershfield, Michael S.

    1979-01-01

    The human lymphoblast line WI-L2 is subject to growth inhibition by a combination of the adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4.) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine. Although adenosine-induced pyrimidine starvation appears to contribute to this effect, uridine only partially reverses adenosine toxicity in WI-L2 and not at all in strain 107, an adenosine kinase-(ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) deficient derivative of WI-L2. Treatment of both cell lines with EHNA and adenosine leads to striking elevations in intracellular S-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methylation reactions. The methylation in vivo of both DNA and RNA is inhibited by concentrations of EHNA and adenosine that elevate intracellular AdoHcy. Addition of 100 μM L-homocysteine thiolactone to cells treated with EHNA and adenosine enhances adenosine toxicity and further elevates AdoHcy to levels approximately 60-fold higher than those obtained in the absence of this amino acid, presumably by combining with adenosine to form AdoHcy in a reaction catalyzed by S-adenosylhomocysteine hydrolase (EC 3.3.1.1). In the adenosine kinase-deficient strain 107, a combination of ADA inhibition and L-homocysteine thiolactone markedly increases intracellular AdoHcy and inhibits growth even in the absence of exogenous adenosine. These results demonstrate a form of toxicity from endogenously produced adenosine and support the view that AdoHcy, by inhibiting methylation, is a mediator of uridine-resistant adenosine toxicity in these human lymphoblast lines. Furthermore, they suggest that AdoHcy may play a role in the pathogenesis of the severe combined immunodeficiency disease found in most children with heritable ADA deficiency. PMID:221926

  18. Role of Adenosine Receptor(s) in the Control of Vascular Tone in the Mouse Pudendal Artery.

    PubMed

    Labazi, Hicham; Tilley, Stephen L; Ledent, Catherine; Mustafa, S Jamal

    2016-03-01

    Activation of adenosine receptors (ARs) has been implicated in the modulation of renal and cardiovascular systems, as well as erectile functions. Recent studies suggest that adenosine-mediated regulation of erectile function is mainly mediated through A2BAR activation. However, no studies have been conducted to determine the contribution of AR subtype in the regulation of the vascular tone of the pudendal artery (PA), the major artery supplying and controlling blood flow to the penis. Our aim was to characterize the contribution of AR subtypes and identify signaling mechanisms involved in adenosine-mediated vascular tone regulation in the PA. We used a DMT wire myograph for muscle tension measurements in isolated PAs from wild-type, A2AAR knockout, A2BAR knockout, and A2A/A2BAR double-knockout mice. Real-time reverse transcription-polymerase chain reaction was used to determine the expression of the AR subtypes. Data from our pharmacologic and genetic approaches suggest that AR activation-mediated vasodilation in the PA is mediated by both the A2AAR and A2BAR, whereas neither the A1AR nor A3AR play a role in vascular tone regulation of the PA. In addition, we showed that A2AAR- and A2BAR-mediated vasorelaxation requires activation of nitric oxide and potassium channels; however, only the A2AAR-mediated response requires protein kinase A activation. Our data are complemented by mRNA expression showing the expression of all AR subtypes with the exception of the A3AR. AR signaling in the PA may play an important role in mediating erection and represent a promising therapeutic option for the treatment of erectile dysfunction. PMID:26718241

  19. Intracellular adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate detection by short-end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor.

    PubMed

    Zinellu, Angelo; Sotgia, Salvatore; Pasciu, Valeria; Madeddu, Manuela; Leoni, Giovanni Giuseppe; Naitana, Salvatore; Deiana, Luca; Carru, Ciriaco

    2008-07-01

    We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test. PMID:18551716

  20. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  1. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  2. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  3. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  4. CD39/Adenosine Pathway Is Involved in AIDS Progression

    PubMed Central

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

    2011-01-01

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

  5. Adenosine receptor modulation of seizure susceptibility in rats

    SciTech Connect

    Szot, P.

    1987-01-01

    Adenosine is considered to be a neuromodulator or cotransmitter in the periphery and CNS. This neuromodulatory action of adenosine may be observed as an anticonvulsant effect. Dose-response curves for R-phenylisopropyladenosine (PIA), cycohexyladenosine (CHA), 2-chloroadenosine (2-ClAdo), N-ethylcarboxamidoadenosine (NECA) and S-PIA were generated against PTZ seizure thresholds in the rat. The rank order of potency for adenosine agonists to elevate PTZ seizure threshold was R-PIA > 2-ClAdo > NECA > CHA > S-PIA. R-PIA was approximately 80-fold more potent than S-PIA. This 80-fold difference in potency between the diasteriomers of PIA was consistent with an A{sub 1} adenoise receptor-mediated response. The anticonvulsant action of 2-ClAdo was reversed by pretreatment with theoplylline. Chronic administration of theophylline significantly increased the specific binding of {sup 3}H-cyclohexyladenosine in membranes of the cerebral cortex and cerebellum of the rat. Chronic exposure to theophylline produced a significant increase in the densities of both the high- and low-affinity forms of A{sub 1} adenosine receptors in the cerebral cortex.

  6. The evolutionary conservation of DNA polymerase. alpha

    SciTech Connect

    Miller, M.A.; Korn, D.; Wang, T.S.F. )

    1988-08-25

    The evolutionary conservation of DNA polymerase {alpha} was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase {alpha} monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase {alpha} catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

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

    PubMed Central

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

    2014-01-01

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

  8. The Rickettsia prowazekii invasion gene homolog (invA) encodes a Nudix hydrolase active on adenosine (5')-pentaphospho-(5')-adenosine.

    PubMed

    Gaywee, Jariyanart; Xu, WenLian; Radulovic, Suzana; Bessman, Maurice J; Azad, Abdu F

    2002-03-01

    The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np(n)N; n > or = 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (5')-pentaphospho-(5')-adenosine into ATP and ADP with a K(m) of 0.1 mM and k(cat) of 1.9 s(-1). Guanosine (5')-pentaphospho-(5')-guanosine and adenosine-(5')-hexaphospho (5')-adenosine are also substrates. Similar to other Nudix hydrolases, InvA requires a divalent metal cation, Mg(2+) or Zn(2+), for optimal activity. These data suggest that the rickettsial invasion protein likely plays a role in controlling the concentration of stress-induced dinucleoside oligophosphates following bacterial invasion.

  9. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    SciTech Connect

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  10. 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.

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

    PubMed Central

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

    2012-01-01

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

  12. 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

  13. Harnessing nature's own cardiac defense mechanism with acadesine, an adenosine regulating agent: importance of the endothelium.

    PubMed

    Engler, R L

    1994-05-01

    Although the effects of adenosine on the heart, including the clinical suppression of cardiac arrhythmias, have been recognized for more than half a century, it is only in the last decade that the therapeutic potential of adenosine has been recognized. Research related to the clinical application of adenosine has concentrated on two areas. The first came directly from early observations about the use of adenosine in treating cardiac arrhythmias, in particular supraventricular tachycardias. The second relates to the use of adenosine to protect the heart from the deleterious consequences of myocardial ischemia and reperfusion. This review will focus on the latter cardioprotective properties of adenosine, particularly those shown by a novel group of drugs termed adenosine regulating agents, the prototype of which is acadesine (Protara).

  14. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats.

    PubMed

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5'-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  15. Gene expression and function of adenosine A(2A) receptor in the rat carotid body.

    PubMed

    Kobayashi, S; Conforti, L; Millhorn, D E

    2000-08-01

    The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A(2A) receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A(2A) and A(2B) Ado receptor mRNAs but not the A(1) or A(3) receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A(2A) receptor mRNA. Immunohistochemical studies further showed that the A(2A) receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca(2+) currents and that this inhibition was abolished by the selective A(2A) receptor antagonist ZM-241385. Ca(2+) imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca(2+)](i). Taken together, our findings indicate that A(2A) receptors are present in type I cells and that activation of A(2A) receptors modulates Ca(2+) accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca(2+) homeostasis and cellular excitability during hypoxia. PMID:10926550

  16. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. PMID:18834537

  17. Flavonoids isolated from Citrus platymamma induced G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells

    PubMed Central

    Nagappan, Arulkumar; Lee, Ho Jeong; Saralamma, Venu Venkatarame Gowda; Park, Hyeon Soo; Hong, Gyeong Eun; Yumnam, Silvia; Raha, Suchismita; Charles, Shobana Nancy; Shin, Sung Chul; Kim, Eun Hee; Lee, Won Sup; Kim, Gon Sup

    2016-01-01

    Citrus platymamma hort. ex Tanaka belongs to the Rutaceae family and is widely used in folk medicines in Korea due to its anti-proliferative, anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. However, the molecular mechanism of its anti-cancer effect is not well understood. The present study was conducted to elucidate the anti-cancer effect and molecular mechanism of flavonoids from Citrus platymamma (FCP) on A549 cells. FCP displayed concentration-dependent inhibition on A549 cells proliferation. Further, flow cytometry revealed that FCP significantly increased the sub-G1 (apoptotic cell population) and G2/M phase population, and the total number of apoptotic cells, in a dose-dependent manner. Nuclear condensation and fragmentation were also observed upon staining with Hoechst 33342 in FCP-treated A549 cells. Immunoblotting demonstrated a dose-dependent downregulation of cyclin B1, cyclin-dependent kinase 1, cell division cycle 25c, pro-caspases −3, −6, −8 and −9, and poly (adenosine diphosphate-ribose) polymerase (PARP) in FCP-treated A549 cells. In addition, FCP induced caspase-3 activation and subsequent PARP cleavage, and increased the B-cell lymphoma (Bcl)-2-associated X protein/Bcl-extra large ratio in A549 cells. These findings suggest that FCP induced G2/M arrest and apoptosis of A549 cells. The present study provides evidence that FCP may be useful in the treatment of human lung cancer. PMID:27446443

  18. Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways.

    PubMed

    Prakobwong, Suksanti; Gupta, Subash C; Kim, Ji Hye; Sung, Bokyung; Pinlaor, Porntip; Hiraku, Yusuke; Wongkham, Sopit; Sripa, Banchob; Pinlaor, Somchai; Aggarwal, Bharat B

    2011-09-01

    Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine(705) and serine(727) and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic

  19. Guggulsterone inhibits human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell growth by inducing caspase-dependent apoptosis and downregulation of survivin and Bcl-2 expression

    PubMed Central

    ZHONG, FEI; YANG, JING; TONG, ZHU-TING; CHEN, LIU-LIU; FAN, LU-LU; WANG, FANG; ZHA, XIA-LI; LI, JUN

    2015-01-01

    Guggulsterone has recently been reported to demonstrate anti-tumor effects in a variety of cancers. The present study aims to investigate the biological roles and underlying mechanism of the action of guggulsterone in cholangiocarcinoma. The immortalized human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell lines were treated with various concentrations of the trans isomer of guggulsterone, Z-guggulsterone. Cellular proliferation was determined using the XTT assay. The apoptotic status of cholangiocarcinoma cells was assessed by Hoechst 33258 staining, DNA fragmentation assay and flow cytometry. Specific caspase inhibitor was used to explore the role of caspase in guggulsterone-induced apoptosis. A colorimetric assay was performed to measure the alterations of the activities of caspase-3, -8 and -9. Western blot analysis was used to detect the protein expression of survivin, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP). As revealed by the present data, guggulsterone significantly inhibited the growth of the two human cholangiocarcinoma cell lines by inducing cellular apoptosis. In addition, guggulsterone-induced apoptosis of cholangiocarcinoma cells was demonstrated to be partially inhibited by the caspase inhibitors z-VAD-fmk, z-LEHD-fmk and z-IETD-fmk, accompanied by the activation of caspases-3, -8 and -9, accumulation of cleaved PARP and decreased expression of survivin and Bcl-2. In conclusion, the present study demonstrated that guggulsterone was able to suppress the proliferation of cholangiocarcinoma by inducing caspase-dependent apoptosis and downregulating survivin and Bcl-2. PMID:26622683

  20. Interruption of the NF-kappaB pathway by Bay 11-7082 promotes UCN-01-mediated mitochondrial dysfunction and apoptosis in human multiple myeloma cells.

    PubMed

    Dai, Yun; Pei, Xin-Yan; Rahmani, Mohamed; Conrad, Daniel H; Dent, Paul; Grant, Steven

    2004-04-01

    Interactions between pharmacologic NF-kappaB inhibitors (eg, Bay 11-7082, SN-50) and the checkpoint abrogator UCN-01 have been examined in human multiple myeloma (MM) cells. Exposure of U266 cells to Bay 11-7082 (Bay) in combination with UCN-01 resulted in the abrogation of NF-kappaB/DNA binding activity and the synergistic induction of apoptosis. Comparable synergism was observed in other MM cell lines and patient-derived CD138+ cells and between an inhibitory peptide of NF-kappaB (SN50) and UCN-01. Bay/UCN-01-mediated lethality involved mitochondrial dysfunction, caspase cleavage, and poly adenosine diphosphate-ribose polymerase (PARP) degradation. Although Bay modestly blocked UCN-01-induced extracellular signal-regulated kinase (ERK) phosphorylation, coadministration activated c-Jun N-terminal kinase (JNK) and cdc2/cdk1 and down-regulated Mcl-1, XIAP, and Bcl-xL. Transfection with a constitutively activated mitogen-activated protein kinase kinase (MEK1)/green fluorescent protein (GFP) construct failed to block apoptosis induced by Bay/UCN-01 but significantly attenuated MEK inhibitor (U0126)/UCN-01-induced lethality. Inhibiting JNK activation with SP600125 or D-JNKI1 peptide markedly reduced Bay/UCN-01-mediated mitochondrial dysfunction and apoptosis and the down-regulation of Mcl-1, XIAP, and Bcl-xL but not of cdc2/cdk1 activation. Stable transfection of cells with dominant-negative caspase-9 dramatically diminished Bay/UCN-01 lethality without altering JNK or cdc2/cdk1 activation. Neither interleukin-6 (IL-6)- nor fibronectin-mediated adherence conferred resistance to Bay/UCN-01-induced apoptosis. Together, these findings suggest that a strategy combining UCN-01 with disruption of the IkappaB kinase (IKK)/IkappaB/NF-kappaB pathway warrants attention in MM.

  1. Reactivity of Cys4 zinc finger domains with gold(III) complexes: insights into the formation of "gold fingers".

    PubMed

    Jacques, Aurélie; Lebrun, Colette; Casini, Angela; Kieffer, Isabelle; Proux, Olivier; Latour, Jean-Marc; Sénèque, Olivier

    2015-04-20

    Gold(I) complexes such as auranofin or aurothiomalate have been used as therapeutic agents for the treatment of rheumatoid arthritis for several decades. Several gold(I) and gold(III) complexes have also shown in vitro anticancer properties against human cancer cell lines, including cell lines resistant to cisplatin. Because of the thiophilicity of gold, cysteine-containing proteins appear as likely targets for gold complexes. Among them, zinc finger proteins have attracted attention and, recently, gold(I) and gold(III) complexes have been shown to inhibit poly(adenosine diphosphate ribose)polymerase-1 (PARP-1), which is an essential protein involved in DNA repair and in cancer resistance to chemotherapies. In this Article, we characterize the reactivity of the gold(III) complex [Au(III)(terpy)Cl]Cl2 (Auterpy) with a model of Zn(Cys)4 "zinc ribbon" zinc finger by a combination of absorption spectroscopy, circular dichroism, mass spectrometry, high-performance liquid chromatography analysis, and X-ray absorption spectroscopy. We show that the Zn(Cys)4 site of Zn·LZR is rapidly oxidized by Auterpy to form a disulfide bond. The Zn(2+) ion is released, and the two remaining cysteines coordinate the Au(+) ion that is produced during the redox reaction. Subsequent oxidation of these cysteines can take place in conditions of excess gold(III) complex. In the presence of excess free thiols mimicking the presence of glutathione in cells, mixing of the zinc finger model and gold(III) complex yields a different product: complex (Au(I))2·LZR with two Au(+) ions bound to cysteines is formed. Thus, on the basis of detailed speciation and kinetic measurements, we demonstrate herein that the destruction of Zn(Cys)4 zinc fingers by gold(III) complexes to achieve the formation of "gold fingers" is worth consideration, either directly or mediated by reducing agents.

  2. Oral treatment with herbal formula B401 alleviates penile toxicity in aging mice with manganism.

    PubMed

    Hsu, Chih-Hsiang; Lin, Ching-Lung; Wang, Sheue-Er; Sheu, Shuenn-Jyi; Chien, Chiang-Ting; Wu, Chung-Hsin

    2015-01-01

    The present study aims to elucidate the roles of nitric oxide synthase activity, oxidative stress, inflammation, and apoptosis in penile toxicity of aging mice associated with excess manganese (Mn) treatment and to investigate the effect of oral treatment with the herbal formula B401 in this respect. ICR strain mice were divided into two groups: the vehicle (sham group) and the B401 (50 mg/kg) group. The mice were orally treated for 5 days; then a high single dose of MnCl2 (100 mg/kg) was given by intraperitoneal injection to the mice. One day after MnCl2 treatment, corpora cavernosal tissues of both Mn-treated mice and their controls were simultaneously sampled to examine their immunohistochemical staining and Western blot analysis. Nitric oxide (NO) production, levels of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), expression levels of factors governing angiogenesis (vascular endothelial growth factor), oxidative stress (catalase, superoxide dismutase 2,4-hydroxynonenal), inflammation (tumor necrosis factor alpha), apoptosis (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein [Bax], cleaved poly(adenosine diphosphate-ribose) polymerase [c-PARP], cytochrome C, caspase-12, and caspase-3) were evaluated in penile corpus cavernosum of the mice. We found that penile toxicity in the mice was enhanced under excess Mn treatment through reduction of NOS activity and increase in oxidative stress, inflammation, and apoptosis in the penile cavernous tissue. Furthermore, the penile toxicity in mice with manganism was alleviated by oral B401 treatment through enhancement of both nitric oxide synthesis and angiogenesis, with simultaneous reduction of oxidative stress, inflammation, and apoptosis in penile corpus cavernosum. We suggest that the herbal formula B401 may serve as a potential dietotherapeutic supplement for penile toxicity or dysfunction in aging males.

  3. Oral treatment with herbal formula B401 alleviates penile toxicity in aging mice with manganism

    PubMed Central

    Hsu, Chih-Hsiang; Lin, Ching-Lung; Wang, Sheue-Er; Sheu, Shuenn-Jyi; Chien, Chiang-Ting; Wu, Chung-Hsin

    2015-01-01

    The present study aims to elucidate the roles of nitric oxide synthase activity, oxidative stress, inflammation, and apoptosis in penile toxicity of aging mice associated with excess manganese (Mn) treatment and to investigate the effect of oral treatment with the herbal formula B401 in this respect. ICR strain mice were divided into two groups: the vehicle (sham group) and the B401 (50 mg/kg) group. The mice were orally treated for 5 days; then a high single dose of MnCl2 (100 mg/kg) was given by intraperitoneal injection to the mice. One day after MnCl2 treatment, corpora cavernosal tissues of both Mn-treated mice and their controls were simultaneously sampled to examine their immunohistochemical staining and Western blot analysis. Nitric oxide (NO) production, levels of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), expression levels of factors governing angiogenesis (vascular endothelial growth factor), oxidative stress (catalase, superoxide dismutase 2,4-hydroxynonenal), inflammation (tumor necrosis factor alpha), apoptosis (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein [Bax], cleaved poly(adenosine diphosphate-ribose) polymerase [c-PARP], cytochrome C, caspase-12, and caspase-3) were evaluated in penile corpus cavernosum of the mice. We found that penile toxicity in the mice was enhanced under excess Mn treatment through reduction of NOS activity and increase in oxidative stress, inflammation, and apoptosis in the penile cavernous tissue. Furthermore, the penile toxicity in mice with manganism was alleviated by oral B401 treatment through enhancement of both nitric oxide synthesis and angiogenesis, with simultaneous reduction of oxidative stress, inflammation, and apoptosis in penile corpus cavernosum. We suggest that the herbal formula B401 may serve as a potential dietotherapeutic supplement for penile toxicity or dysfunction in aging males. PMID:26064043

  4. Potassium increases the antitumor effects of ascorbic acid in breast cancer cell lines in vitro

    PubMed Central

    FRAJESE, GIOVANNI VANNI; BENVENUTO, MONICA; FANTINI, MASSIMO; AMBROSIN, ELENA; SACCHETTI, PAMELA; MASUELLI, LAURA; GIGANTI, MARIA GABRIELLA; MODESTI, ANDREA; BEI, ROBERTO

    2016-01-01

    Ascorbic acid (A) has been demonstrated to exhibit anti-cancer activity in association with chemotherapeutic agents. Potassium (K) is a regulator of cellular proliferation. In the present study, the biological effects of A and K bicarbonate, alone or in combination (A+K), on breast cancer cell lines were evaluated. The survival of cancer cells was determined by sulforhodamine B cell proliferation assay, while analysis of the cell cycle distribution was conducted via fluorescence-activated cell sorting. In addition, the expression of signaling proteins was analyzed upon treatment. The results indicated that there was a heterogeneous response of the different cell lines to A and K, and the best effects were achieved by A+K and A treatment. The interaction between A+K indicated an additive or synergistic effect. In addition, A+K increased the percentage of cells in the sub-G1 phase of the cell cycle, and was the most effective treatment in activating the degradation of poly(adenosine diphosphate-ribose) polymerase-1. In the breast cancer cell line MCF-7, A+K induced the appearance of the 18 kDa isoform of B-cell lymphoma-2-associated X protein (Bax), which is a more potent inducer of apoptosis than the full-length Bax-p21. The effects of A and K on the phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 were heterogeneous. In addition, treatment with K, A and A+K inhibited the expression of nuclear factor-κB. Overall, the results of the present study indicated that K potentiated the anti-tumoral effects of A in breast cancer cells in vitro. PMID:27313770

  5. The Bmi-1 polycomb protein antagonizes the (-)-epigallocatechin-3-gallate-dependent suppression of skin cancer cell survival.

    PubMed

    Balasubramanian, Sivaprakasam; Adhikary, Gautam; Eckert, Richard L

    2010-03-01

    The polycomb group (PcG) proteins are epigenetic regulators of gene expression that enhance cell survival. This regulation is achieved via action of two multiprotein PcG complexes--PRC2 (EED) and PRC1 [B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1)]. These complexes modulate gene expression by increasing histone methylation and reducing acetylation--leading to a closed chromatin conformation. Activity of these proteins is associated with increased cell proliferation and survival. We show increased expression of key PcG proteins in immortalized keratinocytes and skin cancer cell lines. We examine the role of two key PcG proteins, Bmi-1 and enhancer of zeste homolog 2 (Ezh2), and the impact of the active agent in green tea, (-)-epigallocatechin-3-gallate (EGCG), on the function of these regulators. EGCG treatment of SCC-13 cells reduces Bmi-1 and Ezh2 level and this is associated with reduced cell survival. The reduction in survival is associated with a global reduction in histone H3 lysine 27 trimethylation, a hallmark of PRC2 complex action. This change in PcG protein expression is associated with reduced expression of key proteins that enhance progression through the cell cycle [cyclin-dependent kinase (cdk)1, cdk2, cdk4, cyclin D1, cyclin E, cyclin A and cyclin B1] and increased expression of proteins that inhibit cell cycle progression (p21 and p27). Apoptosis is also enhanced, as evidenced by increased caspase 9, 8 and 3 cleavage and increased poly(adenosine diphosphate ribose) polymerase cleavage. EGCG treatment also increases Bax and suppresses Bcl-xL expression. Vector-mediated enhanced Bmi-1 expression reverses these EGCG-dependent changes. These findings suggest that green tea polyphenols reduce skin tumor cell survival by influencing PcG-mediated epigenetic regulatory mechanisms.

  6. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces antiapoptotic and proapoptotic signals in acute myeloid leukemia.

    PubMed

    Faderl, Stefan; Harris, David; Van, Quin; Kantarjian, Hagop M; Talpaz, Moshe; Estrov, Zeev

    2003-07-15

    High levels of cytokines are associated with a poor prognosis in acute myeloid leukemia (AML). However, cytokines may induce, on one hand, survival factor expression and cell proliferation and, on the other hand, expression of inhibitory signals such as up-regulation of suppressors of cytokine signaling (SOCS) and induce apoptotic cell death. Because blasts from patients with AML express high procaspase protein levels, we asked whether granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances procaspase protein production in AML cells. In the GM-CSF-responsive OCIM2 AML cell line, GM-CSF induced signal transducer and activator of transcription 5 (Stat 5) phosphorylation, up-regulated cyclin D2, and stimulated cell cycle progression. Concurrently, GM-CSF stimulated expression of SOCS-2 and -3 and of procaspases 2 and 3 and induced caspase 3 activation, poly(ADP[adenosine 5'-diphosphate]-ribose) polymerase (PARP) cleavage, and apoptotic cell death. The Janus kinase (Jak)-Stat inhibitor AG490 abrogated GM-CSF-induced expression of procaspase 3 and activation of caspase 3. Under the same conditions GM-CSF up-regulated production of BAX as well as Bcl-2, Bcl-XL, survivin, and XIAP. GM-CSF also increased procaspase 3 protein levels in OCI/AML3 and Mo7e cells, suggesting that this phenomenon is not restricted to a single leukemia cell line. Our data suggest that GM-CSF exerts a dual effect: it stimulates cell division but contemporaneously up-regulates Jak-Stat-dependent proapoptotic proteins. Up-regulation of procaspase levels in AML is thus a beacon for an ongoing growth-stimulatory signal.

  7. Celastrol Potentiates Radiotherapy by Impairment of DNA Damage Processing in Human Prostate Cancer

    SciTech Connect

    Dai Yao; DeSano, Jeffrey T.; Meng Yang; J, Qing; Ljungman, Mats; Lawrence, Theodore S.; Xu Liang

    2009-07-15

    Purpose: Celastrol is an active ingredient of traditional herbal medicine and has recently been identified as a potent natural proteasome inhibitor. In the present study, we evaluated the radiosensitizing potential of celastrol in the human prostate cancer PC-3 model. Methods and Materials: Clonogenic assays were performed to determine the radiosensitizing effect of celastrol. Apoptosis was examined by flow cytometry using Annexin V and propidium iodide staining and by a caspase-3 activation assay. DNA damage processing was examined by immunofluorescent staining and Western blot for phosphorylated H2AX ({gamma}H2AX). The PC-3 xenograft model in the athymic nude mouse was used for the determination of the in vivo efficacy of celastrol combined with radiotherapy. The tumor samples were also analyzed for apoptosis and angiogenesis. Results: Celastrol sensitized PC-3 cells to ionizing radiation (IR) in a dose- and schedule-dependent manner, in which pretreatment with celastrol for 1 h followed by IR achieved maximal radiosensitization. Celastrol significantly prolonged the presence of IR-induced {gamma}H2AX and increased IR-induced apoptosis. Celastrol, combined with fractionated radiation, significantly inhibited PC-3 tumor growth in vivo without obvious systemic toxicity. The combination treatment increased {gamma}H2AX levels and apoptosis, induced cleavage of poly(adenosine diphosphate-ribose)polymerase and Mcl-1, and reduced angiogenesis in vivo compared with either treatment alone. Conclusion: Celastrol sensitized PC-3 cells to radiation both in vitro and in vivo by impairing DNA damage processing and augmenting apoptosis. Celastrol might represent a promising new adjuvant regimen for the treatment of hormone-refractory prostate cancer.

  8. Plumbagin-silver nanoparticle formulations enhance the cellular uptake of plumbagin and its antiproliferative activities.

    PubMed

    Appadurai, Prakash; Rathinasamy, Krishnan

    2015-10-01

    Colloidal silver nanoparticles (AgNPs) have attracted much attention in recent years as diagnostics and new drug delivery system in cancer medicine. To study the effects of plumbagin (PLB), a relatively non-toxic napthaquinone isolated from the roots of Plumbago indica in human cervical cancer cell line and developed a formulation to enhance its cytotoxic activities. Silver nanoparticles were synthesised by chemical reduction method and complexed with PLB. Both the AgNPs and the complex PLB-AgNPs were characterised by dynamic light scattering, high-resolution scanning electron microscopy and transmission electron microscopy. The amount of PLB and PLB-AgNPs internalised was determined by ultra-violet-visible spectrophotometer. Cell inhibition was determined by sulphorhodamine B assay. Mitotic index was determined by Wright-Giemsa staining. Apoptosis induction was assessed by western blot using cleaved poly adenosine diphosphate-ribose polymerase antibody. The scanning electron microscope analysis indicated an average particle size of 32±8 nm in diameter. Enhanced internalisation of PLB into the HeLa cells was observed in PLB-AgNPs. PLB inhibited proliferation of cells with IC50 value of about 18±0.6 µM and blocked the cells at mitosis in a concentration-dependent manner. PLB also inhibited the post-drug exposure clonogenic survival of cells and induced apoptosis. The antiproliferative, antimitotic and apoptotic activities were also found to be increased when cells were treated with PLB-AgNPs. The authors results support the idea that AgNP could be a promising and effective drug delivery system for enhanced activity of PLB in cancer treatment. PMID:26435279

  9. Enhanced in vitro antiproliferative effects of EpCAM antibody-functionalized paclitaxel-loaded PLGA nanoparticles in retinoblastoma cells

    PubMed Central

    Mitra, Moutushy; Misra, Ranjita; Harilal, Anju; Sahoo, Sanjeeb K

    2011-01-01

    Background To specifically deliver paclitaxel (PTX) to retinoblastoma (RB) cells, the anionic surface-charged poly(lactic-co-glycolic acid) (PLGA) NPs loaded with paclitaxel were conjugated with epithelial cell adhesion molecule (EpCAM) antibody for enhancing site-specific intracellular delivery of paclitaxel against EpCAM overexpressing RB cells. Methods PTX-loaded PLGA NPs were prepared by the oil-in-water single emulsion solvent evaporation method, and the PTX content in NPs was estimated by the reverse phase isocratic mode of high performance liquid chromatography. Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide chemistry was employed for the covalent attachment of monoclonal EpCAM antibody onto the NP surface. In vitro cytotoxicity of native PTX, unconjugated PTX-loaded NPs (PTX-NPs), and EpCAM antibody-conjugated PTX-loaded nanoparticles (PTX-NP-EpCAM) were evaluated on a Y79 RB cell line by a dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while cellular apoptosis, cysteinyl-aspartic acid protease (caspase)-3 activation, Poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage, and cell-cycle arrest were quantified by flow cytometry. By employing flow cytometry and fluorescence image analyses, the extent of cellular uptake was comparatively evaluated. Results PTX-NP-EpCAM had superior antiproliferation activity, increased arrested cell population at the G2-M phase, and increased activation of caspase-3, followed by PARP cleavage in parallel with the induction of apoptosis. Increased uptake of PTX-Np-EpCAM by the cells suggests that they were mainly taken up through EpCAM mediated endocytosis. Conclusions EpCAM antibody-functionalized biodegradable NPs for tumor-selective drug delivery and overcoming drug resistance could be an efficient therapeutic strategy for retinoblastoma treatment. PMID:22065926

  10. Adenosine Inhibition of Mesopontine Cholinergic Neurons: Implications for EEG Arousal

    PubMed Central

    Rainnie, Donald G.; Grunze, Heinz C. R.; McCarley, Robert W.; Greene, Robert W.

    2013-01-01

    Increased discharge activity of mesopontine cholinergic neurons participates in the production of electroencephalographic (EEG) arousal; such arousal diminishes as a function of the duration of prior wakefulness or of brain hyperthermia. Whole-cell and extracellular recordings in a brainstem slice show that mesopontine cholinergic neurons are under the tonic inhibitory control of endogenous adenosine, a neuromodulator released during brain metabolism. This inhibitory tone is mediated postsynaptically by an inwardly rectifying potassium conductance and by an inhibition of the hyperpolarization-activated current. These data provide a coupling mechanism linking neuronal control of EEG-arousal with the effects of prior wakefulness, brain hyperthermia, and the use of the adenosine receptor blockers caffeine and theophylline. PMID:8303279

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

    PubMed Central

    Festugato, Moira

    2015-01-01

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

  12. Structure–Activity Relationships of 9-Alkyladenine and Ribose-Modified Adenosine Derivatives at Rat A3 Adenosine Receptors†

    PubMed Central

    Jacobson, Kenneth A.; Siddiqi, Suhaib M.; Olah, Mark E.; Ji, Xiao-duo; Melman, Neli; Bellamkonda, Kamala; Meshulam, Yakov; Stiles, Gary L.; Kim, Hea O.

    2012-01-01

    9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5′-(N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3–6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(β-d-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 µM. 2-Chloro-9-[2-amino-2,3-dideoxy-β-d-5-(methylcarbamoyl)-arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3′-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3′-OH and 4′-CH2OH groups of adenosine are not required for activation at A3 receptors. A

  13. Targeting the A2B adenosine receptor during gastrointestinal ischemia and inflammation

    PubMed Central

    Eltzschig, Holger K; Rivera-Nieves, Jesus; Colgan, Sean P

    2014-01-01

    Extracellular adenosine functions as an endogenous distress signal via activation of four distinct adenosine receptors (A1, A2A, A2B and A3). Conditions of limited oxygen availability or acute inflammation lead to elevated levels of extracellular adenosine and enhanced signaling events. This relates to a combination of four mechanisms: i) increased production of adenosine via extracellular phosphohydrolysis of precursor molecules (particularly ATP and ADP); ii) increased expression and signaling via hypoxia-induced adenosine receptors, particularly the A2B adenosine receptor; iii) attenuated uptake from the extracellular towards the intracellular compartment; and iv) attenuated intracellular metabolism. Due to their large surface area, mucosal organs are particularly prone to hypoxia and ischemia associated inflammation. Therefore, it is not surprising that adenosine production and signaling plays a central role in attenuating tissue inflammation and injury during intestinal ischemia or inflammation. In fact, recent studies combining pharmacological and genetic approaches demonstrated that adenosine signaling via the A2B adenosine receptor dampens mucosal inflammation and tissue injury during intestinal ischemia or experimental colitis. This review outlines basic principles of extracellular adenosine production, signaling, uptake and metabolism. In addition, we discuss the role of this pathway in dampening hypoxia-elicited inflammation, specifically in the setting of intestinal ischemia and inflammation. PMID:19769545

  14. Archaeal DNA polymerases in biotechnology.

    PubMed

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Huang, Yanchao

    2015-08-01

    DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-03-15

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

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

    PubMed Central

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

    2016-01-01

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

  19. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

    Ansong, Charles; Ortega, Corrie; Payne, Samuel H.; Haft, Daniel H.; Chauvigne-Hines, Lacie M.; Lewis, Michael P.; Ollodart, Anja R.; Purvine, Samuel O.; Shukla, Anil K.; Fortuin, Suereta; Smith, Richard D.; Adkins, Joshua N.; Grundner, Christoph; Wright, Aaron T.

    2013-01-24

    The annotation of protein function is almost completely performed by in silico approaches. However, computational prediction of protein function is frequently incomplete and error prone. In Mycobacterium tuberculosis (Mtb), ~25% of all genes have no predicted function and are annotated as hypothetical proteins. This lack of functional information severely limits our understanding of Mtb pathogenicity. Current tools for experimental functional annotation are limited and often do not scale to entire protein families. Here, we report a generally applicable chemical biology platform to functionally annotate bacterial proteins by combining activity-based protein profiling (ABPP) and quantitative LC-MS-based proteomics. As an example of this approach for high-throughput protein functional validation and discovery, we experimentally annotate the families of ATP-binding proteins in Mtb. Our data experimentally validate prior in silico predictions of >250 ATPases and adenosine nucleotide-binding proteins, and reveal 73 hypothetical proteins as novel ATP-binding proteins. We identify adenosine cofactor interactions with many hypothetical proteins containing a diversity of unrelated sequences, providing a new and expanded view of adenosine nucleotide binding in Mtb. Furthermore, many of these hypothetical proteins are both unique to Mycobacteria and essential for infection, suggesting specialized functions in mycobacterial physiology and pathogenicity. Thus, we provide a generally applicable approach for high throughput protein function discovery and validation, and highlight several ways in which application of activity-based proteomics data can improve the quality of functional annotations to facilitate novel biological insights.

  20. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Pannell, Lewis K.; Ji, Xiao-duo; Jarvis, Michael F.; Williams, Michael; Hutchison, Alan J.; Barrington, William W.; Stiles, Gary L.

    2011-01-01

    The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology. PMID:2561548

  1. A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

    PubMed

    Wu, Eugene Y; Walsh, Amanda R; Materne, Emma C; Hiltner, Emily P; Zielinski, Bryan; Miller, Bill R; Mawby, Lily; Modeste, Erica; Parish, Carol A; Barnes, Wayne M; Kermekchiev, Milko B

    2015-01-27

    Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5'-to-3' exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E-DNA-ddNTP) and binary (E-DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5'-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.

  2. 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

  3. [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.

  4. Adenosine A1 receptors determine effects of caffeine on total fluid intake but not caffeine appetite.

    PubMed

    Rieg, Timo; Schnermann, Jürgen; Vallon, Volker

    2007-01-26

    Adenosine A1 receptor wild-type (+/+) and knockout (-/-) mice were used to elucidate the role of adenosine A1 receptors in caffeine self-administration in a two-bottle choice test and in the effect of caffeine on total fluid intake and plasma renin concentration. With access to water only, adenosine A1 receptor -/- mice showed greater basal fluid intake and greater plasma renin concentration than +/+ mice. Free access to both water and a caffeinated solution (30 mg/100 ml) for 14 days increased total fluid intake only in adenosine A1 receptor +/+ mice (by 23+/-3%), and both total fluid intake and plasma renin concentration were no longer different between genotypes. Mean intake of water and caffeinated solution was not different between adenosine A1 receptor +/+ and -/- mice. These data reveal that adenosine A1 receptors do not contribute to caffeine consumption, but determine the effects of caffeine on fluid intake and plasma renin concentration. PMID:17126319

  5. Polymerase Gamma Disease through the Ages

    ERIC Educational Resources Information Center

    Saneto, Russell P.; Naviaux, Robert K.

    2010-01-01

    The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 ("POLG"). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems…

  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. Cytoprotective effects of adenosine and inosine in an in vitro model of acute tubular necrosis

    PubMed Central

    Módis, Katalin; Gerő, Domokos; Nagy, Nóra; Szoleczky, Petra; Tóth, Zoltán Dóri; Szabó, Csaba

    2009-01-01

    Background and purpose: We have established an in vitro model of acute tubular necrosis in rat kidney tubular cells, using combined oxygen-glucose deprivation (COGD) and screened a library of 1280 pharmacologically active compounds for cytoprotective effects. Experimental approach: We used in vitro cell-based, high throughput, screening, with cells subjected to COGD using hypoxia chambers, followed by re-oxygenation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the Alamar Blue assay measured mitochondrial respiration and the lactate dehydrogenase assay was used to indicate cell death. ATP levels were measured using a luminometric assay. Key results: Adenosine markedly reduced cellular injury, with maximal cytoprotective effect at 100 µM and an EC50 value of 14 µM. Inosine was also found to be cytoprotective. The selective A3 adenosine receptor antagonist MRS 1523 attenuated the protective effects of adenosine and inosine, while an A3 adenosine receptor agonist provided a partial protective effect. Adenosine deaminase inhibition attenuated the cytoprotective effect of adenosine but not of inosine during COGD. Inhibition of adenosine kinase reduced the protective effects of both adenosine and inosine during COGD. Pretreatment of the cells with adenosine or inosine markedly protected against the fall in cellular ATP content in the cells subjected to COGD. Conclusions and implications: The cytoprotection elicited by adenosine and inosine in a model of renal ischaemia involved both interactions with cell surface adenosine receptors on renal tubular epithelial cells and intracellular metabolism and conversion of adenosine to ATP. PMID:19906119

  8. Adenosine stimulates Ca2+ fluxes and increases cytosolic free Ca2+ in cultured rat mesangial cells.

    PubMed Central

    Olivera, A; López-Rivas, A; López-Novoa, J M

    1992-01-01

    Adenosine has been associated with cellular Ca2+ metabolism in some cell types. Since adenosine is able to contract glomerular mesangial cells in culture, and since Ca2+ is the main messenger mediating contractile responses, we studied the effect of adenosine on 45Ca2+ movements into and out of mesangial cells and on the cytosolic free Ca2+ concentration ([Ca2+]i). Adenosine at 0.1 mM increased 45Ca2+ uptake (basal, 9993 +/- 216; + adenosine, 14823 +/- 410 d.p.m./mg; P less than 0.01) through verapamil-sensitive Ca2+ channels. These channels seem to be of the A1-adenosine receptor subtype. Adenosine also stimulated 45Ca2+ efflux from 45Ca(2+)-loaded mesangial cells. This effect was accompanied by a net depletion of intracellular 45Ca2+ content under isotopic equilibrium conditions (basal, 24213 +/- 978; + adenosine, 18622 +/- 885 d.p.m./mg; P less than 0.05). The increase in 45Ca2+ efflux was inhibited by a Ca(2+)-free medium or in the presence of 10 microM-verapamil. However, the intracellular Ca(2+)-release blocker TMB-8 (10 microM) only partially inhibited the adenosine-stimulated 45Ca2+ efflux. In addition, adenosine induced an elevation in [Ca2+]i in mesangial cells with an initial transient peak within 15 s (basal, 113 +/- 7; adenosine, 345 +/- 46 nM), and a secondary increase which was slower (3-4 min) and of lower magnitude than the initial peak (250 +/- 21 nM). In summary, adenosine elevates [Ca2+]i and stimulates both Ca2+ uptake from the extracellular pool and Ca2+ efflux from intracellular pools in mesangial cells. The Ca2+ release from internal stores is produced by a combination of a TMB-8-inhibitable and a non-TMB-8-inhibitable mechanism, and seems to be dependent on Ca2+ influx. PMID:1554371

  9. A global profile of replicative polymerase usage

    PubMed Central

    Müller, Carolin A.; Miyabe, Izumi; Brooks, Tony; Retkute, Renata; Hubank, Mike; Nieduszyski, Conrad A.; Carr, Antony M.

    2014-01-01

    Three eukaryotic DNA polymerases are essential for genome replication. Polα-primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polε replicates the leading strand while Polδ performs lagging strand synthesis. However, it is not known whether this division of labour is maintained across the whole genome or how uniform it is within single replicons. Using S. pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome–wide. Pu–seq provides direct replication origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labour is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose this results from occasional leading strand initiation by Polδ followed by exchange for Polε. PMID:25664722

  10. Search for New Purine- and Ribose-Modified Adenosine Analogues as Selective Agonists and Antagonists at Adenosine Receptors†

    PubMed Central

    Siddiqi, Suhaib M.; Jacobson, Kenneth A.; Esker, John L.; Olah, Mark E.; Ji, Xiao-duo; Melman, Neli; Tiwari, Kamal N.; Secrist, John A.; Schneller, Stewart W.; Cristalli, Gloria; Stiles, Gary L.; Johnson, Carl R.; IJzerman, Ad P.

    2012-01-01

    The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2′-, 3′-, and 5′-deoxy; 2′- and 3′-O-methyl; 2′-deoxy 2′-fluoro; 6′-thio; 5′-uronamide; carbocyclic; 4′- or 3′-methyl; and inversion of configuration). (−)- and (+)-5′-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6′-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(β-L-2′-deoxy-6′-thiolyxofuranoside) displayed a Ki value of 8 μM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropylxanthine). 2-Chloro-2′-deoxyadenosine and 2-chloroadenin-9-yl(β-D-6′-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 μM, respectively. The A2a selective agonist 2-(1-hexynyl)-5′-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4′-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4′-methyladenosine-5′-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors. PMID:7707320

  11. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  12. 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

  13. Modulation of adenosine signaling prevents scopolamine-induced cognitive impairment in zebrafish.

    PubMed

    Bortolotto, Josiane Woutheres; Melo, Gabriela Madalena de; Cognato, Giana de Paula; Vianna, Mônica Ryff Moreira; Bonan, Carla Denise

    2015-02-01

    Adenosine, a purine ribonucleoside, exhibits neuromodulatory and neuroprotective effects in the brain and is involved in memory formation and cognitive function. Adenosine signaling is mediated by adenosine receptors (A1, A2A, A2B, and A3); in turn, nucleotide and nucleoside-metabolizing enzymes and adenosine transporters regulate its levels. Scopolamine, a muscarinic cholinergic receptor antagonist, has profound amnesic effects in a variety of learning paradigms and has been used to induce cognitive deficits in animal models. This study investigated the effects of acute exposure to caffeine (a non-selective antagonist of adenosine receptors A1 and A2A), ZM 241385 (adenosine receptor A2A antagonist), DPCPX (adenosine receptor A1 antagonist), dipyridamole (inhibitor of nucleoside transporters) and EHNA (inhibitor of adenosine deaminase) in a model of pharmacological cognitive impairment induced by scopolamine in adult zebrafish. Caffeine, ZM 241385, DPCPX, dipyridamole, and EHNA were acutely administered independently via i.p. in zebrafish, followed by exposure to scopolamine dissolved in tank water (200μM). These compounds prevented the scopolamine-induced amnesia without impacting locomotor activity or social interaction. Together, these data support the hypothesis that adenosine signaling may modulate memory processing, suggesting that these compounds present a potential preventive strategy against cognitive impairment.

  14. Impaired inhibitory function of presynaptic A1-adenosine receptors in SHR mesenteric arteries.

    PubMed

    Rocha-Pereira, Carolina; Arribas, Silvia Magdalena; Fresco, Paula; González, Maria Carmen; Gonçalves, Jorge; Diniz, Carmen

    2013-01-01

    In hypertension, vascular reactivity alterations have been attributed to numerous factors, including higher sympathetic innervation/adenosine. This study examined the modulation of adenosine receptors on vascular sympathetic nerves and their putative contribution to higher noradrenaline spillover in hypertension. We assessed adenosine receptors distribution in the adventitia through confocal microscopy, histomorphometry, and their regulatory function on electrically-evoked [(3)H]-noradrenaline overflow, using selective agonists/antagonists. We found that: i) A1-adenosine receptor agonist (CPA: 100 nM) inhibited tritium overflow to a lower extent in SHR (25% ± 3%, n = 14) compared to WKY (38% ± 3%, n = 14) mesenteric arteries; ii) A2A-adenosine receptor agonist (CGS 21680: 100 nM) induced a slight increase of tritium overflow that was similar in SHR (22% ± 8%, n = 8) and WKY (24% ± 5%, n = 8) mesenteric arteries; iii) A2B- and A3-adenosine receptors did not alter tritium overflow in either strain; iv) all adenosine receptors were present on mesenteric artery sympathetic nerves and/or some adventitial cells of both strains; and v) A1-adenosine receptor staining fractional area was lower in SHR than in WKY mesenteric arteries. We conclude that there is an impaired inhibitory function of vascular presynaptic A1-adenosine receptors in SHR, likely related to a reduced presence of these receptors on sympathetic innervation, which might lead to higher levels of noradrenaline in the synaptic cleft and contribute to hypertension in this strain.

  15. Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

    PubMed Central

    Kelley, G G; Aassar, O S; Forrest, J N

    1991-01-01

    The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport. Images PMID:1752953

  16. Characterization and regulation of adenosine transport in T84 intestinal epithelial cells.

    PubMed

    Mun, E C; Tally, K J; Matthews, J B

    1998-02-01

    Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl- secretion. Previous data suggest that A2b receptor-mediated Cl- secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na(+)-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5'-(N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl- secretory responses and hence diarrheal disorders.

  17. Thermally multiplexed polymerase chain reaction.

    PubMed

    Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

    2015-07-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  18. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  19. Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions.

    PubMed

    Kashio, Makiko; Sokabe, Takaaki; Shintaku, Kenji; Uematsu, Takayuki; Fukuta, Naomi; Kobayashi, Noritada; Mori, Yasuo; Tominaga, Makoto

    2012-04-24

    The ability to sense temperature is essential for organism survival and efficient metabolism. Body temperatures profoundly affect many physiological functions, including immunity. Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca(2+)-permeable cation channel expressed in a wide range of immunocytes. TRPM2 is activated by adenosine diphosphate ribose and hydrogen peroxide (H(2)O(2)), although the activation mechanism by H(2)O(2) is not well understood. Here we report a unique activation mechanism in which H(2)O(2) lowers the temperature threshold for TRPM2 activation, termed "sensitization," through Met oxidation and adenosine diphosphate ribose production. This sensitization is completely abolished by a single mutation at Met-214, indicating that the temperature threshold of TRPM2 activation is regulated by redox signals that enable channel activity at physiological body temperatures. Loss of TRPM2 attenuates zymosan-evoked macrophage functions, including cytokine release and fever-enhanced phagocytic activity. These findings suggest that redox signals sensitize TRPM2 downstream of NADPH oxidase activity and make TRPM2 active at physiological body temperature, leading to increased cytosolic Ca(2+) concentrations. Our results suggest that TRPM2 sensitization plays important roles in macrophage functions.

  20. Use of RNA polymerase molecular beacon assay to measure RNA polymerase interactions with model promoter fragments.

    PubMed

    Mekler, Vladimir; Severinov, Konstantin

    2015-01-01

    RNA polymerase-promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase-promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase-promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase-promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.

  1. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  2. Adenosine triphosphate stress echocardiography in the detection of myocardial ischemia.

    PubMed

    Fukai, T; Koyanagi, S; Tashiro, H; Ichiki, T; Tsutsui, H; Matsumoto, T; Takeshita, A

    1995-10-01

    The purpose of this study was to assess feasibility and safety in the diagnosis of coronary artery in the diagnosis of coronary artery disease and myocardial ischemia using adenosine triphosphate (ATP) stress echocardiography. ATP, a product of human myocardial tissue, is more potent than adenosine in increasing coronary blood flow. Like adenosine, ATP also has a short half-life (<10 s). Left ventricular echocardiograms were recorded during step-wise infusions of ATP in 86 patients who underwent coronary angiography and stress thallium 201 scintigraphy. No serious complications occurred with ATP infusion and most of the side effects were mild and transient. Significant coronary artery disease (>75% diameter stenosis) was present in 34 of 48 patients who had normal echocardiograms at rest. The sensitivity and specificity of ATP-induced wall motion abnormalities for coronary artery disease was 65% (22 of 34) and 100% (14 of 14), respectively. The sensitivity was 50% (10 of 20) in those with one-vessel disease and 86% (12 of 14) in those with multivessel disease (P < .05). In patients with normal echocardiograms at rest and without prior myocardial infarction, the sensitivity of ATP stress echocardiography for the detection of myocardial ischemia assessed by 201Tl single proton emission computed tomography was 58%, with a specificity of 76%, and a diagnostic accuracy of 66%. The sensitivity was 43% in those with one-vessel disease, and 86% in those with multivessel disease (P = .05). In patients with prior myocardial infarction, the sensitivity of ATP stress echocardiography for the detection of viable but jeopardized myocardium was 81%, with a specificity of 91%. The patients with well-developed collateral circulation had a higher incidence of developing wall motion abnormality than those without collaterals (70% v 40%, P < .01). ATP stress echocardiography is valuable for the assessment of coronary artery disease in patients with multivessel disease, coronary

  3. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  4. Diminished adenosine A1 receptor expression on macrophages in brain and blood of patients with multiple sclerosis.

    PubMed

    Johnston, J B; Silva, C; Gonzalez, G; Holden, J; Warren, K G; Metz, L M; Power, C

    2001-05-01

    The nucleoside adenosine has been shown to control the production of proinflammatory molecules through its actions on cell surface purine receptors. Previously, we have reported that the adenosine A1 receptor (A1AR) regulates tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) expression and exhibits diminished function in patients with multiple sclerosis (MS; Mayne et al., Ann Neurol 1999;45:633-639). In the present study, A1AR expression in both brain and peripheral blood mononuclear cells (PBMC) from MS and control groups was characterized by fluorescence-activated cell sorting (FACS), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical analyses. FACS analyses of PBMC revealed that A1AR expression was chiefly detectable on CD14-positive cells and was reduced by 53.1% (p < 0.01) in MS patients compared to controls. A1AR mRNA levels were reduced by 43.1% (p < 0.001) in the brains of MS patients compared to patients with other neurological diseases and controls. A1AR protein expression in brain was detected primarily in CD45-positive glial cells and was markedly diminished in MS patients. The analysis of A1AR transcripts in the brain revealed that the A1AR-beta transcript was diminished (49.2%) in MS patients compared to controls (p < 0.002). These results indicate that the A1AR, expressed principally on cells of monocyte/macrophage lineage in both brain and blood, is selectively diminished in MS patients. Reduction of the A1AR-beta transcript in MS patients suggests that dysregulated splicing may influence A1AR protein levels, potentially leading to increased macrophage activation and central nervous system inflammation. PMID:11357956

  5. 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.

  6. The Role of Uridine Adenosine Tetraphosphate in the Vascular System

    PubMed Central

    Matsumoto, Takayuki; Tostes, Rita C.; Webb, R. Clinton

    2011-01-01

    The endothelium plays a pivotal role in vascular homeostasis, and endothelial dysfunction is a major feature of cardiovascular diseases, such as arterial hypertension, atherosclerosis, and diabetes. Recently, uridine adenosine tetraphosphate (Up4A) has been identified as a novel and potent endothelium-derived contracting factor (EDCF). Up4A structurally contains both purine and pyrimidine moieties, which activate purinergic receptors. There is an accumulating body of evidence to show that Up4A modulates vascular function by actions on endothelial and smooth muscle cells. In this paper, we discuss the effects of Up4A on vascular function and a potential role for Up4A in cardiovascular diseases. PMID:22110488

  7. Vasodilatory responsiveness to adenosine triphosphate in ageing humans

    PubMed Central

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

    2010-01-01

    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

  8. Adenosine conjugated lipidic nanoparticles for enhanced tumor targeting.

    PubMed

    Swami, Rajan; Singh, Indu; Jeengar, Manish Kumar; Naidu, V G M; Khan, Wahid; Sistla, Ramakrishna

    2015-01-01

    Delivering chemotherapeutics by nanoparticles into tumor is impeded majorly by two factors: nonspecific targeting and inefficient penetration. Targeted delivery of anti-cancer agents solely to tumor cells introduces a smart strategy because it enhances the therapeutic index compared with untargeted drugs. The present study was performed to investigate the efficiency of adenosine (ADN) to target solid lipid nanoparticles (SLN) to over expressing adenosine receptor cell lines such as human breast cancer and prostate cancer (MCF-7 and DU-145 cells), respectively. SLN were prepared by emulsification and solvent evaporation process using docetaxel (DTX) as drug and were characterized by various techniques like dynamic light scattering, differential scanning calorimeter and transmission electron microscopy. DTX loaded SLNs were surface modified with ADN, an adenosine receptors ligand using carbodiimide coupling. Conjugation was confirmed using infrared spectroscopy and quantified using phenol-sulfuric acid method. Conjugated SLN were shown to have sustained drug release as compared to unconjugated nanoparticles and drug suspension. Compared with free DTX and unconjugated SLN, ADN conjugated SLN showed significantly higher cytotoxicity of loaded DTX, as evidenced by in vitro cell experiments. The IC50 was 0.41 μg/ml for native DTX, 0.30 μg/ml for unconjugated SLN formulation, and 0.09 μg/ml for ADN conjugated SLN formulation in MCF-7 cell lines. Whereas, in DU-145, there was 2 fold change in IC50 of ADN-SLN as compared to DTX. IC50 was found to be 0.44 μg/ml for free DTX, 0.39 μg/ml for unconjugated SLN and 0.22 μg/ml for ADN-SLN. Annexin assay and cell cycle analysis assay further substantiated the cell cytotoxicity. Fluorescent cell uptake and competitive ligand-receptor binding assay corroborated the receptor mediated endocytosis pathway indicated role of adenosine receptors in internalization of conjugated particles. Pharmacokinetic studies of lipidic

  9. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses

    PubMed Central

    2014-01-01

    Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages. PMID:25105011

  10. Adenosine Inhibits the Excitatory Synaptic Inputs to Basal Forebrain Cholinergic, GABAergic, and Parvalbumin Neurons in Mice

    PubMed Central

    Yang, Chun; Franciosi, Serena; Brown, Ritchie E.

    2013-01-01

    Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal forebrain (BF) region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV) neurons to determine the effect of adenosine. Whole-cell recordings were made from BF cholinergic neurons and from BF GABAergic and PV neurons with the size (>20 μm) and intrinsic membrane properties (prominent H-currents) corresponding to cortically projecting neurons. A brief (2 min) bath application of adenosine (100 μM) decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (EPSCs) in all groups of BF cholinergic, GABAergic, and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1 μM). Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1 receptor-mediated inhibition of glutamatergic inputs to cortically projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required

  11. An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep

    PubMed Central

    Bjorness, Theresa E.; Dale, Nicholas; Mettlach, Gabriel; Sonneborn, Alex; Sahin, Bogachan; Fienberg, Allen A.; Yanagisawa, Masashi; Bibb, James A.

    2016-01-01

    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. SIGNIFICANCE STATEMENT 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

  12. Presynaptic action of adenosine on a 4-aminopyridine-sensitive current in the rat carotid body

    PubMed Central

    Vandier, C; Conway, A F; Landauer, R C; Kumar, P

    1999-01-01

    Plasma adenosine concentration increases during hypoxia to a level that excites carotid body chemoreceptors by an undetermined mechanism. We have examined this further by determining the electrophysiological responses to exogenous adenosine of sinus nerve chemoafferents in vitro and of whole-cell currents in isolated type I cells.Steady-state, single-fibre chemoafferent discharge was increased approximately 5-fold above basal levels by 100 μM adenosine. This adenosine-stimulated discharge was reversibly and increasingly reduced by methoxyverapamil (D600, 100 μM), by application of nickel chloride (Ni2+, 2 mM) and by removal of extracellular Ca2+. These effects strongly suggest a presynaptic, excitatory action of adenosine on type I cells of the carotid body.Adenosine decreased whole-cell outward currents at membrane potentials above -40 mV in isolated type I cells recorded during superfusion with bicarbonate-buffered saline solution at 34–36 °C. This effect was reversible and concentration dependent with a maximal effect at 10 μM.The degree of current inhibition induced by 10 μM adenosine was voltage independent (45.39 ± 2.55% (mean ± s.e.m.) between −40 and +30 mV) and largely (∼75%), but not entirely, Ca2+ independent. 4-Aminopyridine (4-AP, 5 mM) decreased the amplitude of the control outward current by 80.60 ± 3.67% and abolished the effect of adenosine.Adenosine was without effect upon currents near the resting membrane potential of approximately −55 mV and did not induce depolarization in current-clamp experiments.We conclude that adenosine acts to inhibit a 4-AP-sensitive current in isolated type I cells of the rat carotid body and suggest that this mechanism contributes to the chemoexcitatory effect of adenosine in the whole carotid body. PMID:10050009

  13. Ribonucleic Acid Polymerase in Allomyces arbuscula

    PubMed Central

    Cain, Alice K.; Nester, Eugene W.

    1973-01-01

    Three distinct species of ribonucleic acid (RNA) polymerase were resolved from Allomyces arbuscula by diethylaminoethyl-cellulose chromatography and characterized as to ionic strength and divalent cation preference. α-Amanitin specifically inhibited enzyme II; neither rifampin nor cycloheximide had any effect on the three enzymes. RNA polymerase was isolated from three stages of the diploid life cycle: the hyphal growth stage, mycelia in the process of forming sporangia, and the mitospores. The same three enzyme species could be resolved from each stage. Thus, there is no evidence from this work that RNA polymerase plays a major role in the control of development. PMID:4728272

  14. Adenosine 3', 5'-cyclic monophosphate levels in Thermomonospora curvata during cellulase biosynthesis

    SciTech Connect

    Fennington, G.; Neubauer, D.; Stutzenberger, F.

    1983-01-01

    The enzymatic degradation of cellulose requires the synergistic activity of at least three enzymes: exo-beta-1,4-glucanase (EC3.2.1.91), endo-beta-1,4-glucanase (EC3.2.1.4), and beta-glucosidase (EC3.2.1.21). Despite extensive studies on a variety of cellulolytic bacteria and fungi, the mechanism(s) regulating the biosynthesis of this inducible catabolic enzyme complex remains unknown. The intracellular concentrations of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate (cAMP) have been shown to play a major role in mediating catabolite repression of enzyme biosynthesis. The cAMP acts through a cAMP receptor protein (termed CRP or CAP) which is a dimer having two identical subunits each capable of binding one molecule of cAMP. The N-terminal domain of the CRP binds the cAMP while the C-terminal domain binds to DNA at the promotor region of a cAMP-dependent operon and stimulates transcription by promoting the formation of a preinitiation complex between RNA polymerase and the DNA. Intracellular cAMP levels in E. coli (the prototype organism for such studies) are influenced by the type and availability of carbon source used for growth. High intracellular cAMP levels should lead to higher concentrations of cAMP-CRP complexes which should increase the transcription rates for cAMP-dependent operons (such as the lac operon of beta-galactosidase) and indeed the differential rate of beta-galactosidase biosynthesis correlates to intracellular cAMP levels. In the case of cellulase, catabolite repression by glucose or other readily metabolizable compounds closely controls production in an apparently similar manner and therefore a correlation may exist between enzyme biosynthesis and intracellular cAMP levels. This communication describes the fluctuation in cAMP levels during cellulase induction and repression in the thermophilic actinomycete, Thermomonospora curvata.

  15. Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

    PubMed Central

    Shi, Xing-Xing; Yin, Bai-Shuang; Yang, Peng; Chen, Hao; Li, Xin; Su, Li-Xue; Fan, Hong-Gang; Wang, Hong-Bin

    2016-01-01

    Xylazine is a potent analgesic extensively used in veterinary and animal experimentation. Evidence exists that the analgesic effect can be inhibited using adenosine 5’-monophosphate activated protein kinase (AMPK) inhibitors. Considering this idea, the aim of this study was to investigate whether the AMPK signaling pathway is involved in the central analgesic mechanism of xylazine in the rat. Xylazine was administrated via the intraperitoneal route. Sprague-Dawley rats were sacrificed and the cerebral cortex, cerebellum, hippocampus, thalamus and brainstem were collected for determination of liver kinase B1 (LKB1) and AMPKα mRNA expression using quantitative real-time polymerase chain reaction (qPCR), and phosphorylated LKB1 and AMPKα levels using western blot. The results of our study showed that compared with the control group, xylazine induced significant increases in AMPK activity in the cerebral cortex, hippocampus, thalamus and cerebellum after rats received xylazine (P < 0.01). Increased AMPK activities were accompanied with increased phosphorylation levels of LKB1 in corresponding regions of rats. The protein levels of phosphorylated LKB1 and AMPKα in these regions returned or tended to return to control group levels. However, in the brainstem, phosphorylated LKB1 and AMPKα protein levels were decreased by xylazine compared with the control (P < 0.05). In conclusion, our data indicates that xylazine alters the activities of LKB1 and AMPK in the central nervous system of rats, which suggests that xylazine affects the regulatory signaling pathway of the analgesic mechanism in the rat brain. PMID:27049320

  16. Effect of pulsed electromagnetic field exposure on adenosine receptors in rat brain.

    PubMed

    Varani, Katia; Vincenzi, Fabrizio; Targa, Martina; Corciulo, Carmen; Fini, Milena; Setti, Stefania; Cadossi, Ruggero; Borea, Pier Andrea

    2012-05-01

    Different effects of pulsed electromagnetic field (PEMF) exposure on brain tissue have been described in pre-clinical models and in clinical settings. Nevertheless, the mechanism of action and the possible interaction with membrane receptors such as adenosine receptors (ARs) has not been investigated. The present study focused on the effect of PEMFs on A1 and A2A ARs in the rat cerebral cortex and cortical neurons. Affinity and density of ARs were evaluated by means of saturation binding experiments while mRNA expression was investigated through retro-transcription polymerase chain reaction (RT-PCR). PEMF treatment of the intact rat cerebral cortex or cortical neurons at 1.5 mT mediated a transient and significant increase in A2A ARs after 4 h (2.0-fold increase) and 6 h (1.4- and 1.8-fold increase, respectively) of exposure. In addition, PEMF treatment of the rat cerebral cortex and rat cortical neurons at 3 mT upregulated A2A ARs after 2 h (2.0- and 2.2-fold increase, respectively) and 4 h (1.6- and 1.9-fold increase, respectively). The treatment of rat cortex membranes with PEMFs at 1.5 and 3 mT induced an increase in A2A AR density after 2 h (1.9- and 2.2-fold increase, respectively) and was constant at all incubation times investigated. In rat cortical neurons, mRNA levels of A1 and A2A ARs were not affected by PEMF exposure for the times and intensities used. These results suggest that PEMF treatment has different biological effects in whole organs or cells in comparison with isolated membranes.

  17. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  18. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  19. Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes

    PubMed Central

    Bolcato, Chiara; Cusan, Claudia; Pastorin, Giorgia; Cacciari, Barbara; Klotz, Karl Norbert; Morizzo, Erika

    2007-01-01

    In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach. PMID:18368532

  20. Exploring human adenosine A3 receptor complementarity and activity for adenosine analogues modified in the ribose and purine moiety

    PubMed Central

    Van Rompaey, Philippe; Jacobson, Kenneth A.; Gross, Ariel S.; Gao, Zhan-Guo; Van Calenbergh, Serge

    2012-01-01

    In this paper we investigated the influence on affinity, selectivity and intrinsic activity upon modification of the adenosine agonist scaffold at the 3′- and 5′-positions of the ribofuranosyl moiety and the 2- and N6-positions of the purine base. This resulted in the synthesis of various analogues, that is, 3–12 and 24–33, with good hA3AR selectivity and moderate-to-high affinities (as in 32, Ki = 27 nM). Interesting was the ability to tune the intrinsic activity depending on the substituent introduced at the 3′-position. PMID:15670905

  1. Structures of the Leishmania infantum polymerase beta

    PubMed Central

    Mejia, Edison; Burak, Matthew; Alonso, Ana; Larraga, Vicente; Kunkel, Thomas A.; Bebenek, Katarzyna; Garcia-Diaz, Miguel

    2014-01-01

    Protozoans of the genus Leishmania, the pathogenic agent causing leishmaniasis, encode the family X DNA polymerase Li Pol β. Here, we report the first crystal structures of Li Pol β. Our pre- and post-catalytic structures show that the polymerase adopts the common family X DNA polymerase fold. However, in contrast to other family X DNA polymerases, the dNTP-induced conformational changes in Li Pol β are much more subtle. Moreover, pre- and post-catalytic structures reveal that Li Pol β interacts with the template strand through a nonconserved, variable region known as loop3. Li Pol β Δloop3 mutants display a higher catalytic rate, catalytic efficiency and overall error rates with respect to WT Li Pol β. These results further demonstrate the subtle structural variability that exists within this family of enzymes and provides insight into how this variability underlies the substantial functional differences among their members. PMID:24666693

  2. Opiate-induced changes in brain adenosine levels and narcotic drug responses.

    PubMed

    Wu, M; Sahbaie, P; Zheng, M; Lobato, R; Boison, D; Clark, J D; Peltz, G

    2013-01-01

    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 twofold 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 receptor(1) (A(1)) agonist (2-chloro-N6-cyclopentyladenosine, 0.5mg/kg) or an A(2a) receptor (A(2a)) antagonist (SCH 58261, 1mg/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.

  3. Photomodulation of G Protein-Coupled Adenosine Receptors by a Novel Light-Switchable Ligand

    PubMed Central

    2015-01-01

    The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities. PMID:25248077

  4. Perfusion pressure control by adenosine triphosphate given during cardiopulmonary bypass.

    PubMed

    Hashimoto, K; Kurosawa, H; Horikoshi, S; Miyamoto, H; Suzuki, K

    1993-01-01

    Administration of exogenous adenosine triphosphate (ATP) as a vasodilator during cardiopulmonary bypass was assessed in consecutive adult patients (n = 24) who demonstrated a high arterial perfusion pressure (mean, > 90 mm Hg). The action of ATP was characterized by rapid induction and stabilization of the blood pressure level. The dose of ATP ranged from 0.68 to 2.68 mg/min. Within 1 minute after the administration, there was a significant reduction in the perfusion pressure from 102 +/- 18 mm Hg (mean +/- standard deviation) to 72 +/- 19 mm Hg. The ATP was then able to maintain the desired pressure of 69 +/- 12 mm Hg at 5 minutes, 67 +/- 12 mm Hg at 10 minutes, and consistent values thereafter. After the ATP administration was discontinued, there was a prompt recovery of pressure without bradyarrhythmia. The frequency and amount of inotropes used were consistent with the control group (n = 26). Although the administration of ATP reduced the increase in serum catecholamine concentration, there were no significant changes in other vasoactive mediators (eicosanoid, angiotensin II, endothelin) between the two groups during cardiopulmonary bypass. There was neither an accumulation of metabolic products (uric acid, phosphate) nor a decrease in the level of divalent cation (Ca2+), which is observed when the cations combine with phosphates or adenosine nucleotides. This study confirmed the efficacy and safety of ATP infusion during cardiopulmonary bypass. PMID:8417658

  5. 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. PMID:25957126

  6. Novel trypanocidal analogs of 5'-(methylthio)-adenosine.

    PubMed

    Sufrin, Janice R; Spiess, Arthur J; Marasco, Canio J; Rattendi, Donna; Bacchi, Cyrus J

    2008-01-01

    The purine nucleoside 5'-deoxy-5'-(hydroxyethylthio)-adenosine (HETA) is an analog of the polyamine pathway metabolite 5'-deoxy-5'-(methylthio)-adenosine (MTA). HETA is a lead structure for the ongoing development of selectively targeted trypanocidal agents. Thirteen novel HETA analogs were synthesized and examined for their in vitro trypanocidal activities against bloodstream forms of Trypanosoma brucei brucei LAB 110 EATRO and at least one drug-resistant Trypanosoma brucei rhodesiense clinical isolate. New compounds were also assessed in a cell-free assay for their activities as substrates of trypanosome MTA phosphorylase. The most potent analog in this group was 5'-deoxy-5'-(hydroxyethylthio)-tubercidin, whose in vitro cytotoxicity (50% inhibitory concentration [IC50], 10 nM) is 45 times greater than that of HETA (IC50, 450 nM) against pentamidine-resistant clinical isolate KETRI 269. Structure-activity analyses indicate that the enzymatic cleavage of HETA analogs by trypanosome MTA phosphorylase is not an absolute requirement for trypanocidal activity. This suggests that additional biochemical mechanisms are associated with the trypanocidal effects of HETA and its analogs.

  7. Purification and Properties of Adenosine Diphosphoglucose Pyrophosphorylase from Sweet Corn 1

    PubMed Central

    Amir, Jacob; Cherry, Joe H.

    1972-01-01

    A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg2+ and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1-phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 × 10−4, 3.2 × 10−5, 3.3 × 10−5, and 6.2 × 10−4m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 × 10−7m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate. PMID:16658078

  8. Adenosine and ATP Link PCO2 to Cortical Excitability via pH

    PubMed Central

    Dulla, Chris G.; Dobelis, Peter; Pearson, Tim; Frenguelli, Bruno G.; Staley, Kevin J.; Masino, Susan A.

    2007-01-01

    Summary In addition to affecting respiration and vascular tone, deviations from normal CO2 alter pH, consciousness, and seizure propensity. Outside the brainstem, however, the mechanisms by which CO2 levels modify neuronal function are unknown. In the hippocampal slice preparation, increasing CO2, and thus decreasing pH, increased the extracellular concentration of the endogenous neuromodulator adenosine and inhibited excitatory synaptic transmission. These effects involve adenosine A1 and ATP receptors and depend on decreased extracellular pH. In contrast, decreasing CO2 levels reduced extracellular adenosine concentration and increased neuronal excitability via adenosine A1 receptors, ATP receptors, and ecto-ATPase. Based on these studies, we propose that CO2-induced changes in neuronal function arise from a pH-dependent modulation of adenosine and ATP levels. These findings demonstrate a mechanism for the bidirectional effects of CO2 on neuronal excitability in the forebrain. PMID:16364904

  9. Respiratory stimulant effects of adenosine in man after caffeine and enprofylline.

    PubMed Central

    Smits, P; Schouten, J; Thien, T

    1987-01-01

    In a double-blind and randomized study the respiratory stimulant effect of continuous intravenous adenosine infusion was studied after previous administration of caffeine, placebo and enprofylline in 10 healthy young volunteers. After placebo, adenosine induced an increase of minute ventilation (from 6.3 to 12.5 l min-1), tidal volume (from 0.60 to 0.96 l), and breathing rate (from 11.0 to 14.8 min-1). Venous pCO2 fell and pH rose after adenosine. Caffeine significantly reduced the adenosine-induced changes of minute ventilation, tidal volume, venous pCO2 and pH, whereas no changes occurred after enprofylline. Our results suggest that adenosine stimulates respiration in man by binding with specific P1-purinoceptors, which can be blocked by caffeine, but not by enprofylline. PMID:3440102

  10. Reconstruction of the adenosine system by bone marrow-derived mesenchymal stem cell transplantation☆

    PubMed Central

    Kang, Huicong; Hu, Qi; Liu, Xiaoyan; Liu, Yinhe; Xu, Feng; Li, Xiang; Zhu, Suiqiang

    2012-01-01

    In the present study, we transplanted bone marrow-derived mesenchymal stem cells into the CA3 area of the hippocampus of chronic epilepsy rats kindled by lithium chloride-pilocarpine. Immunofluorescence and western blotting revealed an increase in adenosine A1 receptor expression and a decrease in adenosine A2a receptor expression in the brain tissues of epileptic rats 3 months after transplantation. Moreover, the imbalance in the A1 adenosine receptor/A2a adenosine receptor ratio was improved. Electroencephalograms showed that frequency and amplitude of spikes in the hippocampus and frontal lobe were reduced. These results suggested that mesenchymal stem cell transplantation can reconstruct the normal function of the adenosine system in the brain and greatly improve epileptiform discharges. PMID:25806064

  11. 2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

    PubMed

    Konno, Takashi; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-08-22

    The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production.

  12. Involvement of adenosine A2a receptor in intraocular pressure decrease induced by 2-(1-octyn-1-yl)adenosine or 2-(6-cyano-1-hexyn-1-yl)adenosine.

    PubMed

    Konno, Takashi; Murakami, Akira; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-04-01

    The aim of the present study is to clarify the mechanism for the decrease in intraocular pressure by 2-alkynyladenosine derivatives in rabbits. The receptor binding analysis revealed that 2-(1-octyn-1-yl)adenosine (2-O-Ado) and 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) selectively bound to the A(2a) receptor with a high affinity. Ocular hypotensive responses to 2-O-Ado and 2-CN-Ado were inhibited by the adenosine A(2a)-receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), but not by the adenosine A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the adenosine A(2b)-receptor antagonist alloxazine. In addition, 2-O-Ado and 2-CN-Ado caused an increase in outflow facility, which was inhibited by CSC, but not by DPCPX or alloxazine. Moreover, 2-O-Ado and 2-CN-Ado increased cAMP in the aqueous humor, and the 2-O-Ado-induced an increase in cAMP was inhibited by CSC. These results suggest that 2-O-Ado and 2-CN-Ado reduced intraocular pressure via an increase in outflow facility. The ocular hypotension may be mainly mediated through the activation of adenosine A(2a) receptor, although a possible involvement of adenosine A(1) receptor cannot be completely ruled out. 2-O-Ado and 2-CN-Ado are useful lead compounds for the treatment of glaucoma.

  13. Real-time monitoring of extracellular adenosine using enzyme-linked microelectrode arrays.

    PubMed

    Hinzman, Jason M; Gibson, Justin L; Tackla, Ryan D; Costello, Mark S; Burmeister, Jason J; Quintero, Jorge E; Gerhardt, Greg A; Hartings, Jed A

    2015-12-15

    Throughout the central nervous system extracellular adenosine serves important neuroprotective and neuromodulatory functions. However, current understanding of the in vivo regulation and effects of adenosine is limited by the spatial and temporal resolution of available measurement techniques. Here, we describe an enzyme-linked microelectrode array (MEA) with high spatial (7500 µm(2)) and temporal (4 Hz) resolution that can selectively measure extracellular adenosine through the use of self-referenced coating scheme that accounts for interfering substances and the enzymatic breakdown products of adenosine. In vitro, the MEAs selectively measured adenosine in a linear fashion (r(2)=0.98±0.01, concentration range=0-15 µM, limit of detection =0.96±0.5 µM). In vivo the limit of detection was 0.04±0.02 µM, which permitted real-time monitoring of the basal extracellular concentration in rat cerebral cortex (4.3±1.5 µM). Local cortical injection of adenosine through a micropipette produced dose-dependent transient increases in the measured extracellular concentration (200 nL: 6.8±1.8 µM; 400 nL: 19.4±5.3 µM) [P<0.001]. Lastly, local injection of dipyridamole, which inhibits transport of adenosine through equilibrative nucleoside transporter, raised the measured extracellular concentration of adenosine by 120% (5.6→12.3 µM) [P<0.001]. These studies demonstrate that MEAs can selectively measure adenosine on temporal and spatial scales relevant to adenosine signaling and regulation in normal and pathologic states. PMID:26183072

  14. Adenosine regulates the proinflammatory signaling function of thrombin in endothelial cells

    PubMed Central

    Hassanian, Seyed Mahdi; Dinarvand, Peyman; Rezaie, Alireza R.

    2014-01-01

    The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B>A2A>A1>A3) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A-specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1 and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1 and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. PMID:24477600

  15. Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism

    PubMed Central

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

    2010-01-01

    Introduction Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility. Aim The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism. Methods Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol–modified ADA (PEG–ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG–ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation. Main Outcome Measures Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays. Results We found that lowering adenosine levels in penile tissues by PEG–ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG–ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism. Conclusions Our studies have identified that PEG–ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation

  16. Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis

    PubMed Central

    Gaudin, Alice; Lepetre-Mouelhi, Sinda; Mougin, Julie; Parrod, Martine; Pieters, Grégory; Garcia-Argote, Sébastien; Loreau, Olivier; Goncalves, Jordan; Chacun, Hélène; Courbebaisse, Yann; Clayette, Pascal; Desmaële, Didier; Rousseau, Bernard; Andrieux, Karine; Couvreur, Patrick

    2015-01-01

    Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[3H]-Adenosine NAs and [14C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1 h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury. PMID:26087468

  17. Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis.

    PubMed

    Gaudin, Alice; Lepetre-Mouelhi, Sinda; Mougin, Julie; Parrod, Martine; Pieters, Grégory; Garcia-Argote, Sébastien; Loreau, Olivier; Goncalves, Jordan; Chacun, Hélène; Courbebaisse, Yann; Clayette, Pascal; Desmaële, Didier; Rousseau, Bernard; Andrieux, Karine; Couvreur, Patrick

    2015-08-28

    Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury. PMID:26087468

  18. Effect of adenosine and adenosine receptor antagonist on Müller cell potassium channel in Rat chronic ocular hypertension models.

    PubMed

    Yang, Zijian; Huang, Ping; Liu, Xiaohong; Huang, Shouyue; Deng, Lianfu; Jin, Zhe; Xu, Shuo; Shen, Xi; Luo, Xunda; Zhong, Yisheng

    2015-01-01

    Müller cells are principal glial cells in rat retina and have attracted much attention in glaucoma studies. However, it is not clear whether adenosine and adenosine receptor (AR) antagonists play any roles in the regulation of potassium channels in Müller cells and subsequently in the promotion of glutamine synthetase (GS) and L-Glutamate/L-Aspartate Transporter (GLAST) functions. We found that chronic ocular hypertension (COH) in rat down-regulated Müller cells Kir2.1, Kir4.1, TASK-1, GS and GLAST expressions and attenuated the peak of inward potassium current. Retinal ganglion cells (RGC) count was lower in the COH rats than that in the sham operation animals. Intravitreal injection of selective A2A AR antagonist SCH442416 up-regulated Müller cell Kir4.1, TASK-1, GS and GLAST expressions and enhanced inward potassium currents compared with those in the COH rats with vehicle control. Meanwhile, the RGC count was higher following intravitreal injection of SCH442416 in the COH rats than that after vehicle injection. The fact that PKA inhibitor H-89 blocked these SCH442416 effects suggested that the PKA signaling pathway was involved in the observed ocular responses following the intravitreal SCH442416 injection. PMID:26063641

  19. Spectroscopic and theoretical investigations of adenosine 5'-diphosphate and adenosine 5'-triphosphate dianions in the gas phase.

    PubMed

    Schinle, Florian; Crider, Paul E; Vonderach, Matthias; Weis, Patrick; Hampe, Oliver; Kappes, Manfred M

    2013-05-14

    Doubly deprotonated adenosine 5'-diphosphate ([ADP-2H](2-)) and adenosine 5'-triphosphate ([ATP-2H](2-)) dianions were investigated using infrared multiple photon dissociation (IR-MPD) and photoelectron spectroscopy. Vibrational spectra acquired in the X-H stretch region (X = C, N, O) and augmented by isotope-labelling were compared to density functional theory (DFT) calculations at the B3LYP/TZVPP level. This suggests that in [ATP-2H](2-) the two phosphate groups adjacent to the ribose ring are preferentially deprotonated. Photoelectron spectra recorded at 4.66 and 6.42 eV photon energies revealed adiabatic detachment energies of 1.35 eV for [ADP-2H](2-) and 3.35 eV for [ATP-2H](2-). Repulsive Coulomb barriers were estimated at ~2.2 eV for [ADP-2H](2-) and ~1.9 eV for [ATP-2H](2-). Time-dependent DFT calculations have been used to simulate the photoelectron spectra. Photodetachment occurs primarily from lone pair orbitals on oxygen atoms within the phosphate chain. PMID:23258289

  20. A role for adenosine in coronary vasoregulation in man. Effects of theophylline and enprofylline.

    PubMed

    Edlund, A; Conradsson, T; Sollevi, A

    1995-11-01

    Adenosine has been suggested to have a role in regulation of the tone of the cardiac resistance vessels. To elucidate the coronary vasoregulatory role of endogenous adenosine in man, we studied the effects of adenosine receptor antagonism by theophylline on coronary blood flow at rest and during light exercise. However, theophylline may also exert pharmacological effects not related to adenosine antagonism. To clarify the contribution of endogenous adenosine in coronary hyperaemia, the effect of theophylline was compared to that of enprofylline, a xanthine which exerts similar pharmacological effects as theophylline while lacking antagonistic action at adenosine receptors. Twenty healthy subjects (10 males) aged 22-39 years were examined. Coronary sinus (CS) blood flow and blood oxygen content were determined at rest and during supine bicycle exercise, at a load of 50 watts, for 10 min. Thereafter, stepwise infusion of adenosine (30 to 60 micrograms/kg/min into the subclavian vein) was performed. Theophylline or enprofylline treatment was instituted randomly and double-blind (10 in each group), and the procedures (i.e. determinations at rest, during exercise and during infusion of adenosine) were repeated. In all 20 subjects, basal CS flow was 70 +/- 6 ml/min and the cardiac oxygen extraction ((A-CS)O2D) was 123 +/- 3 ml/l. During exercise, CS flow and (A-CS)O2D increased to 135 +/- 17 ml/min and 132 +/- 3 ml/l, respectively. Adenosine increased CS flow dose dependently to 161 +/- 27 ml/min, while (A-CS)O2D decreased to 66 +/- 7 ml/l. The vasodilatory effect of adenosine was readily counteracted by theophylline, the increase in CS flow being 33% vs. 133% in the control situation. Enprofylline, on the other hand, enhanced the response to exogenous adenosine. Theophylline, at a dose lacking effect on heart rate and blood pressure, decreased CS flow at rest by 14% (P < 0.05) and during exercise by 18% (P < 0.05). ((A-CS)O2D increased by 14% at rest and during exercise

  1. New high fidelity polymerases from Thermococcus species.

    PubMed

    Griffiths, Kate; Nayak, Sunil; Park, Kyusung; Mandelman, David; Modrell, Brett; Lee, Jun; Ng, Bernie; Gibbs, Moreland D; Bergquist, Peter L

    2007-03-01

    Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability. PMID:16982200

  2. N6-adenosine methylation in MiRNAs.

    PubMed

    Berulava, Tea; Rahmann, Sven; Rademacher, Katrin; Klein-Hitpass, Ludgar; Horsthemke, Bernhard

    2015-01-01

    Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression. PMID:25723394

  3. N6-Adenosine Methylation in MiRNAs

    PubMed Central

    Berulava, Tea; Rahmann, Sven; Rademacher, Katrin; Klein-Hitpass, Ludgar; Horsthemke, Bernhard

    2015-01-01

    Methylation of N6-adenosine (m6A) has been observed in many different classes of RNA, but its prevalence in microRNAs (miRNAs) has not yet been studied. Here we show that a knockdown of the m6A demethylase FTO affects the steady-state levels of several miRNAs. Moreover, RNA immunoprecipitation with an anti-m6A-antibody followed by RNA-seq revealed that a significant fraction of miRNAs contains m6A. By motif searches we have discovered consensus sequences discriminating between methylated and unmethylated miRNAs. The epigenetic modification of an epigenetic modifier as described here adds a new layer to the complexity of the posttranscriptional regulation of gene expression. PMID:25723394

  4. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

    Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

    2009-01-01

    A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

  5. Development and structural analysis of adenosine site binding tankyrase inhibitors.

    PubMed

    Haikarainen, Teemu; Waaler, Jo; Ignatev, Alexander; Nkizinkiko, Yves; Venkannagari, Harikanth; Obaji, Ezeogo; Krauss, Stefan; Lehtiö, Lari

    2016-01-15

    Tankyrases 1 and 2, the specialized members of the ARTD protein family, are druggable biotargets whose inhibition may have therapeutic potential against cancer, metabolic disease, fibrotic disease, fibrotic wound healing and HSV viral infections. We have previously identified a novel tankyrase inhibitor scaffold, JW55, and showed that it reduces mouse colon adenoma formation in vivo. Here we expanded the scaffold and profiled the selectivity of the compounds against a panel of human ARTDs. The scaffold also enables a fine modulation of selectivity towards either tankyrase 1 or tankyrase 2. In order to get insight about the binding mode of the inhibitors, we solved crystal structures of the compounds in complex with tankyrase 2. The compounds bind to the adenosine pocket of the catalytic domain and cause changes in the protein structure that are modulated by the chemical modifications of the compounds. The structural analysis allows further rational development of this compound class as a potent and selective tankyrase inhibitor. PMID:26706174

  6. Development and structural analysis of adenosine site binding tankyrase inhibitors.

    PubMed

    Haikarainen, Teemu; Waaler, Jo; Ignatev, Alexander; Nkizinkiko, Yves; Venkannagari, Harikanth; Obaji, Ezeogo; Krauss, Stefan; Lehtiö, Lari

    2016-01-15

    Tankyrases 1 and 2, the specialized members of the ARTD protein family, are druggable biotargets whose inhibition may have therapeutic potential against cancer, metabolic disease, fibrotic disease, fibrotic wound healing and HSV viral infections. We have previously identified a novel tankyrase inhibitor scaffold, JW55, and showed that it reduces mouse colon adenoma formation in vivo. Here we expanded the scaffold and profiled the selectivity of the compounds against a panel of human ARTDs. The scaffold also enables a fine modulation of selectivity towards either tankyrase 1 or tankyrase 2. In order to get insight about the binding mode of the inhibitors, we solved crystal structures of the compounds in complex with tankyrase 2. The compounds bind to the adenosine pocket of the catalytic domain and cause changes in the protein structure that are modulated by the chemical modifications of the compounds. The structural analysis allows further rational development of this compound class as a potent and selective tankyrase inhibitor.

  7. Extraction and analysis of adenosine triphosphate from aquatic environments

    USGS Publications Warehouse

    Stephens, Doyle W.; Shultz, David J.

    1981-01-01

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

  8. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

    PubMed

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  9. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity

    PubMed Central

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  10. Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate.

    PubMed

    Woody, A Y; Vader, C R; Woody, R W; Haley, B E

    1984-06-19

    A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.

  11. Adenosine, Ketogenic Diet and Epilepsy: The Emerging Therapeutic Relationship Between Metabolism and Brain Activity

    PubMed Central

    Masino, S.A; Kawamura, M; Wasser, C.D.; Pomeroy, L.T; Ruskin, D.N

    2009-01-01

    For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a “retaliatory metabolite.” As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor–based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed. PMID:20190967

  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.

  13. Adenosine dry powder inhalation for bronchial challenge testing, part 2: proof of concept in asthmatic subjects.

    PubMed

    Lexmond, Anne J; van der Wiel, Erica; Hagedoorn, Paul; Bult, Wouter; Frijlink, Henderik W; ten Hacken, Nick H T; de Boer, Anne H

    2014-09-01

    Adenosine is an indirect stimulus to assess bronchial hyperresponsiveness (BHR(2)) in asthma. Bronchial challenge tests are usually performed with nebulised solutions of adenosine 5'-monophosphate (AMP(3)). The nebulised AMP test has several disadvantages, like long administration times and a restrictive maximum concentration that does not result in BHR in all patients. In this study, we investigated the applicability of dry powder adenosine for assessment of BHR in comparison to nebulised AMP. Dry powder adenosine was prepared in doubling doses (0.01-80 mg) derived from the nebulised AMP test with addition of two higher doses. Five asthmatic subjects performed two bronchial challenge tests, one with nebulised AMP following the 2-min tidal breathing method; the second with dry powder adenosine administered with an investigational inhaler and single slow inhalations (inspiratory flow rate 30-40 L/min). All subjects reached a 20% fall in FEV₁(4) with the new adenosine test (PD20(5)) compared to four subjects with the AMP test (PC₂₀(6)). Dry powder adenosine was well tolerated by all subjects and better appreciated than nebulised AMP. In conclusion, this new bronchial challenge test appears to be a safe and convenient alternative to the nebulised AMP test to assess BHR in asthmatic subjects.

  14. Adenosine, ketogenic diet and epilepsy: the emerging therapeutic relationship between metabolism and brain activity.

    PubMed

    Masino, S A; Kawamura, M; Wasser, C D; Wasser, C A; Pomeroy, L T; Ruskin, D N

    2009-09-01

    For many years the neuromodulator adenosine has been recognized as an endogenous anticonvulsant molecule and termed a "retaliatory metabolite." As the core molecule of ATP, adenosine forms a unique link between cell energy and neuronal excitability. In parallel, a ketogenic (high-fat, low-carbohydrate) diet is a metabolic therapy that influences neuronal activity significantly, and ketogenic diets have been used successfully to treat medically-refractory epilepsy, particularly in children, for decades. To date the key neural mechanisms underlying the success of dietary therapy are unclear, hindering development of analogous pharmacological solutions. Similarly, adenosine receptor-based therapies for epilepsy and myriad other disorders remain elusive. In this review we explore the physiological regulation of adenosine as an anticonvulsant strategy and suggest a critical role for adenosine in the success of ketogenic diet therapy for epilepsy. While the current focus is on the regulation of adenosine, ketogenic metabolism and epilepsy, the therapeutic implications extend to acute and chronic neurological disorders as diverse as brain injury, inflammatory and neuropathic pain, autism and hyperdopaminergic disorders. Emerging evidence for broad clinical relevance of the metabolic regulation of adenosine will be discussed. PMID:20190967

  15. Evidence for an antagonism between caffeine and adenosine in the human cardiovascular system.

    PubMed

    Smits, P; Boekema, P; De Abreu, R; Thien, T; van 't Laar, A

    1987-08-01

    A randomized, double-blind and placebo-controlled study was performed in 10 normotensive male subjects to analyze a possible antagonism between caffeine and adenosine with respect to their effects on the cardiovascular system in humans. Caffeine alone, 250 mg intravenously (i.v.), increased blood pressure by 9/12 mm Hg, and resulted in a fall of heart rate (HR) of 3 beats/min. Plasma epinephrine (E) rose by 114% after caffeine. Adenosine alone, in an increasing dose of 0.04-0.16 mg/kg/min, induced an increase in systolic blood pressure (SBP) (17 mm Hg), and HR (33 beats/min), a moderate fall in diastolic blood pressure (DBP) (-4 mm Hg), and no change of mean arterial pressure (MAP). At the highest adenosine infusion rate, forearm blood flow, skin temperature (ST), and transcutaneous oxygen tension were lowered, whereas plasma (nor)epinephrine was increased 227.2 and 215.9%, respectively. Adenosine infusion after caffeine induced comparable effects, but the fractional adenosine-induced changes of SBP, HR, plasma catecholamines, plasma renin activity (PRA), and aldosterone all were significantly reduced by previous administration of caffeine. Our results indicate an antagonism between caffeine and adenosine in humans, which may support the suggestion that some circulatory effects of caffeine are caused by an interaction with endogenous adenosine. PMID:2441163

  16. Luciferase-based assay for adenosine: application to S-adenosyl-L-homocysteine hydrolase.

    PubMed

    Burgos, Emmanuel S; Gulab, Shivali A; Cassera, María B; Schramm, Vern L

    2012-04-17

    S-Adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (K(m), k(cat)) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10(-7) unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.

  17. Role of adenosine signalling and metabolism in β-cell regeneration

    SciTech Connect

    Andersson, Olov

    2014-02-01

    Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?.

  18. The resurgence of A2B adenosine receptor signaling

    PubMed Central

    Aherne, Carol M.; Kewley, Emily M.; Eltzschig, Holger K.

    2010-01-01

    Since its discovery as a low-affinity adenosine receptor (AR), the A2B receptor (A2BAR), has proven enigmatic in its function. The previous discovery of the A2AAR, which shares many similarities with the A2BAR but demonstrates significantly greater affinity for its endogenous ligand, led to the original perception that the A2BAR was not of substantial physiologic relevance. In addition, lack of specific pharmacological agents targeting the A2BAR made its initial characterization challenging. However, the importance of this receptor was reconsidered when it was observed that the A2BAR is highly transcriptionally regulated by factors implicated in inflammatory hypoxia. Moreover, the notion that during ischemia or inflammation extracellular adenosine is dramatically elevated to levels sufficient for A2BAR activation, indicated that A2BAR signaling may be important to dampen inflammation particularly during tissue hypoxia. In addition, the recent advent of techniques for murine genetic manipulation along with development of pharmacological agents with enhanced A2BAR specificity has provided invaluable tools for focused studies on the explicit role of A2BAR signaling in different disease models. Currently, studies performed with combined genetic and pharmacological approaches have demonstrated that A2BAR signaling plays a tissue protective role in many models of acute diseases e.g. myocardial ischemia, or acute lung injury. These studies indicate that the A2BAR is expressed on a wide variety of cell types and exerts tissue/cell specific effects. This is an important consideration for future studies where tissue or cell type specific targeting of the A2BAR may be used as therapeutic approach. PMID:20546702

  19. Structural and Metabolic Specificity of Methylthiocoformycin for Malarial Adenosine Deaminases

    SciTech Connect

    Ho, M.; Cassera, M; Madrid, D; Ting, L; Tyler, P; Kim, K; Almo, S; Schramm, V

    2009-01-01

    Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5?-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5?-methylthioribosyl groups are rotated 130 degrees. A hydrogen bonding network between Asp172 and the 3?-hydroxyl of MT-coformycin is essential for recognition of the 5?-methylthioribosyl group. Water occupies the 5?-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.

  20. Amp Synthesis in Aqueous Solution of Adenosine and Phosphorus Pentoxide

    NASA Astrophysics Data System (ADS)

    Yamagata, Y.; Kojima, H.; Ejiri, K.; Inomata, K.

    1982-12-01

    Possible formation of a P4O10 molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2'-AMP, 3'-AMP, 5'-AMP, cyclic (2' 3')-AMP and cyclic (3' 5')-AMP. The main components of the products were 2'- and 3'-AMP, though cyclic (2' 3')-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl2, 0.01 M MgCl2), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl2, 0.01 M MgCl2), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl2, 0.01 M MgCl2), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).

  1. DNA polymerase alpha and beta in the California urchin.

    PubMed Central

    Racine, F M; Morris, P W

    1978-01-01

    DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species. PMID:569291

  2. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

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

    PubMed Central

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

    2015-01-01

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

  4. Adenosine modulates light responses of rat retinal ganglion cell photoreceptors througha cAMP-mediated pathway

    PubMed Central

    Sodhi, Puneet; Hartwick, Andrew T E

    2014-01-01

    Adenosine is an established neuromodulator in the mammalian retina, with A1 adenosine receptors being especially prevalent in the innermost ganglion cell layer. Activation of A1 receptors causes inhibition of adenylate cyclase, decreases in intracellular cyclic AMP (cAMP) levels and inhibition of protein kinase A (PKA). In this work, our aim was to characterize the effects of adenosine on the light responses of intrinsically photosensitive retinal ganglion cells (ipRGCs) and to determine whether these photoreceptors are subject to neuromodulation through intracellular cAMP-related signalling pathways. Using multielectrode array recordings from postnatal and adult rat retinas, we demonstrated that adenosine significantly shortened the duration of ipRGC photoresponses and reduced the number of light-evoked spikes fired by these neurons. The effects were A1 adenosine receptor-mediated, and the expression of this receptor on melanopsin-containing ipRGCs was confirmed by calcium imaging experiments on isolated cells in purified cultures. While inhibition of the cAMP/PKA pathway by adenosine shortened ipRGC light responses, stimulation of this pathway with compounds such as forskolin had the opposite effect and lengthened the duration of ipRGC spiking. Our findings reveal that the modification of ipRGC photoresponses through a cAMP/PKA pathway is a general feature of rat ganglion cell photoreceptors, and this pathway can be inhibited through activation of A1 receptors by adenosine. As adenosine levels in the retina rise at night, adenosinergic modulation of ipRGCs may serve as an internal regulatory mechanism to limit transmission of nocturnal photic signals by ipRGCs to the brain. Targeting retinal A1 adenosine receptors for ipRGC inhibition represents a potential therapeutic target for sleep disorders and migraine-associated photophobia. PMID:25038240

  5. Role of adenosine signaling on pentylenetetrazole-induced seizures in zebrafish.

    PubMed

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

    2015-04-01

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

  6. Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

    PubMed

    Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2014-09-15

    Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists.

  7. Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

    PubMed

    Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2014-09-15

    Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists. PMID:25063794

  8. Adenosine Modulates the Oocyte Developmental Competence by Exposing Stages and Synthetic Blocking during In Vitro Maturation.

    PubMed

    Cheon, Yong-Pil

    2016-06-01

    Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.. PMID:27660830

  9. 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

  10. Adenosine Modulates the Oocyte Developmental Competence by Exposing Stages and Synthetic Blocking during In Vitro Maturation

    PubMed Central

    Cheon, Yong-Pil

    2016-01-01

    Purine metabolism is known factor for nuclear maturation of oocytes through both follicle cells and oocyte itself. However, it is largely unknown the roles of purine metabolism in the oocyte competence for fertilization and early development. In this study, the effects of adenosine in oocyte competence for development were examined using adenosine and its synthetic inhibitors. Adenosine treatment from GV intact stage for 18 hr (fGV) caused of decrease the fertilization rate but of increase the cleavage rate compared from the other stage treatment groups. Hadacidin did not effect on fertilization rate but increased cleavage rate without stage specificity. Adenosine did not block the effects of hadacidin with the exception of fGV group. By the inhibition of purine synthetic pathways the fertilization rate was decreased in the fGV and fGVB groups but increased in the fMII group. Exogenous adenosine caused of decrease fertilization rate in the fGVB group but increase in the fMII group. Cleavage rate was dramatically increased in the adenosine treatment with synthetic inhibitors. It means that the metabolism of purine has stage specific effects on fertilization and cleavage. Exogenous adenosine had only can improve oocyte developmental competence when it treated at GV intact stage. On the other hand, endogenous synthesis in all maturation stage caused of increase the cleavage rate without effects on fertilization. These data suggest that adenosine at GV stage as a paracrine fashion and inhibitions of endogenous adenosine in all stage improve oocyte developmental competence.. PMID:27660830

  11. Smoke extract impairs adenosine wound healing: implications of smoke-generated reactive oxygen species.

    PubMed

    Allen-Gipson, Diane S; Zimmerman, Matthew C; Zhang, Hui; Castellanos, Glenda; O'Malley, Jennifer K; Alvarez-Ramirez, Horacio; Kharbanda, Kusum; Sisson, Joseph H; Wyatt, Todd A

    2013-05-01

    Adenosine concentrations are elevated in the lungs of patients with asthma and chronic obstructive pulmonary disease, where it balances between tissue repair and excessive airway remodeling. We previously demonstrated that the activation of the adenosine A2A receptor promotes epithelial wound closure. However, the mechanism by which adenosine-mediated wound healing occurs after cigarette smoke exposure has not been investigated. The present study investigates whether cigarette smoke exposure alters adenosine-mediated reparative properties via its ability to induce a shift in the oxidant/antioxidant balance. Using an in vitro wounding model, bronchial epithelial cells were exposed to 5% cigarette smoke extract, were wounded, and were then stimulated with either 10 μM adenosine or the specific A2A receptor agonist, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA; 10 μM), and assessed for wound closure. In a subset of experiments, bronchial epithelial cells were infected with adenovirus vectors encoding human superoxide dismutase and/or catalase or control vector. In the presence of 5% smoke extract, significant delay was evident in both adenosine-mediated and CPCA-mediated wound closure. However, cells pretreated with N-acetylcysteine (NAC), a nonspecific antioxidant, reversed smoke extract-mediated inhibition. We found that cells overexpressing mitochondrial catalase repealed the smoke extract inhibition of CPCA-stimulated wound closure, whereas superoxide dismutase overexpression exerted no effect. Kinase experiments revealed that smoke extract significantly reduced the A2A-mediated activation of cyclic adenosine monophosphate-dependent protein kinase. However, pretreatment with NAC reversed this effect. In conclusion, our data suggest that cigarette smoke exposure impairs A2A-stimulated wound repair via a reactive oxygen species-dependent mechanism, thereby providing a better understanding of adenosine signaling that may direct the development of pharmacological

  12. Vasodilator effects of adenosine on retinal arterioles in streptozotocin-induced diabetic rats.

    PubMed

    Nakazawa, Taisuke; Mori, Asami; Saito, Maki; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2008-02-01

    Adenosine is a potent vasodilator of retinal blood vessels and is implicated to be a major regulator of retinal blood flow during metabolic stress, but little is known about the impact of diabetes on the role of adenosine in regulation of retinal hemodynamics. Therefore, we examined how diabetes affects adenosine-induced vasodilation of retinal arterioles. Male Wistar rats were treated with streptozotocin (80 mg/kg, intraperitoneally), and experiments were performed 6-8 weeks later. Rats were treated with tetrodotoxin (50 microg/kg, intravenously [i.v.]) to eliminate any nerve activity and prevent movement of the eye and infused with methoxamine continuously to maintain adequate systemic circulation. Fundus images were captured with a digital camera that was equipped with a special objective lens, and diameters of retinal arterioles were measured. Adenosine increased diameters of retinal arterioles and decreased systemic blood pressure. These responses were significantly attenuated by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (30 mg/kg, i.v.) and the adenosine triphosphate-dependent K+ (K(ATP)) channel blocker glibenclamide (20 mg/kg, i.v.). The depressor responses to adenosine were reduced in diabetic rats, whereas diabetes did not alter vasodilation of retinal arterioles to adenosine. In contrast, both depressor response and vasodilation of retinal arteriole to acetylcholine were reduced in diabetic rats. The retinal vasodilator responses to adenosine and acetylcholine observed in diabetic rats were diminished by N(G)-nitro-L-arginine methyl ester. There were no differences in the responses to pinacidil, a K(ATP) channel opener, between the diabetic and nondiabetic rats. These results suggest that both the activation of nitric oxide synthase and opening of K(ATP) channels contribute to the vasodilator effects of adenosine in rats in vivo. However, diabetes has no significant impact on the vasodilation mediated by these mechanisms in

  13. [RNA polymerase as a molecular machine].

    PubMed

    Spirin, A S

    2002-01-01

    Structure and function of DNA-dependent RNA polymerase is considered in terms of a conveying molecular machine. The use of mechanical energy and mechanical devices, such as "power-stroke motor", is supposed unlikely in the conveying function of RNA polymerase, as well as other molecular machines. Brownian motion and thermal mobility of macromolecules and their parts are postulated as the only motive impulse at the molecular level. Binding of substrates and subsequent chemical reaction as the energy input may provide successive selection and fixation of alternative conformational states of the enzyme complex thus providing the directionality of the conveyance ("Brownian ratchet mechanism"). The following sequence of events "substrate binding--fixation of a certain conformational state--chemical reaction--fixation of an alternative conformational state--translocation (dissociation and downstream reassociation) of product-template duplex" is proposed as the principal scheme of the forward movement of RNA polymerase along DNA template.

  14. Binding of adenosine and receptor-specific analogues to lymphocytes from control subjects and patients with common variable immunodeficiency.

    PubMed Central

    Shah, T; Simpson, R J; Webster, A D; Peters, T J

    1987-01-01

    Studies were performed on the binding of tritiated adenosine and its analogues, 5'-N-ethylcarboxamide adenosine (NECA) and N6-phenylisopropyladenosine (PIA), to human peripheral blood lymphocytes. These revealed binding only of adenosine (Kd, 1-10 microM, 14,000 binding sites/cell), which was abolished by dipyridamole, a specific adenosine transport inhibitor, suggesting that the binding is to the nucleoside transporter. The absence of high affinity (Kd less than or equal to 1 microM) binding of adenosine or of the two analogues. NECA and PIA suggests that the previously reported effects of adenosine on cAMP formation are not mediated by cell surface specific nucleoside receptors. Binding of adenosine to the carrier in lymphocytes from patients with common variable immunodeficiency was similar to those from control subjects. PMID:2958197

  15. Development of a luminescent G-quadruplex-selective iridium(III) complex for the label-free detection of adenosine

    PubMed Central

    Lu, Lihua; Zhong, Hai-Jing; He, Bingyong; Leung, Chung-Hang; Ma, Dik-Lung

    2016-01-01

    A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R2 = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis. PMID:26778273

  16. Development of a luminescent G-quadruplex-selective iridium(III) complex for the label-free detection of adenosine

    NASA Astrophysics Data System (ADS)

    Lu, Lihua; Zhong, Hai-Jing; He, Bingyong; Leung, Chung-Hang; Ma, Dik-Lung

    2016-01-01

    A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R2 = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis.

  17. DNA sequence polymorphism within the bovine adenosine monophosphate deaminase 1 (AMPD1) is associated with production traits in Chinese cattle.

    PubMed

    Wei, C-B; Wang, J-Q; Chen, F-Y; Niu, H; Li, K

    2015-02-06

    The objectives of the present study were to detect an 18-bp deletion mutation in the bovine adenosine monophosphate deaminase 1 (AMPD1) gene and analyze its effect on growth traits in 2 Chinese cattle breeds using DNA sequencing and agarose electrophoresis. The five 19-bp polymerase chain reaction products of the AMPD1 gene exhibited 3 genotypes and 2 alleles: WW: homozygote genotype (wild-type); DD: homozygote genotype (mutant-type); WD: heterozygote genotype. Frequencies of the W allele varied from 66.15-70.35%. The associations between the 18-bp deletion mutation in the AMPD1 gene with production traits in 226 Jia-Xian red cattle was analyzed. The animals with genotype WW showed significantly higher heart girth and body weight than those with genotypes WD and DD at 24 months (P < 0.01). Our results indicate that the deletion mutation in the AMPD1 gene is associated with production traits, and may be used for marker-assisted selection in beef cattle breeding programs.

  18. A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Bustamante, Salvador; García-Sacristán, Albino; Orensanz, Luis M

    1999-01-01

    The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. In U46619 (10−7  M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10−6  M), adenosine and related analogues induced relaxations with the following potency order: 5′-N-ethylcarboxamidoadenosine (NECA)=5′-(N-cyclopropyl)-carboxamidoadenosine (CPCA)=2-chloroadenosine (2-CA)>adenosine>cyclopentyladenosine (CPA)=N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA)=2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680). Epithelium removal or incubation with indomethacin (3×10−6  M) and L-NG-nitroarginine (L-NOARG, 3×10−5  M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10−8 M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3×10−8  M and 10−7  M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10−5  M) and DPCPX (10−6  M), which block A1/A2-receptors, reduced such relaxations. In strips treated with guanethidine (10−5  M), atropine (10−7  M), L-NOARG (3×10−5  M) and indomethacin (3×10−6  M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10−4  M) induced contractions of preparations. 8-PT (10−5  M) increased both contractions. DPCPX (10−8  M), NECA (10−4  M), CPCA, (10−4  M) and 2-CA (10−4  M) did not alter the contractions to EFS. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids

  19. Design, Synthesis, and Identification of 4″α-Azidoethyl-cyclic ADP-Carbocyclic-ribose as a Highly Potent Analogue of Cyclic ADP-Ribose, a Ca(2+)-Mobilizing Second Messenger.

    PubMed

    Sato, Takatoshi; Watanabe, Mizuki; Tsuzuki, Takayoshi; Takano, Satoshi; Murayama, Takashi; Sakurai, Takashi; Kameda, Tomoshi; Fukuda, Hayato; Arisawa, Mitsuhiro; Shuto, Satoshi

    2016-08-11

    Cyclic adenosine diphosphate-carbocyclic-ribose (cADPcR, 2) is a stable equivalent of cyclic adenosine diphosphate-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. On the basis of the structure-activity relationship of cADPR-related compounds and three-dimensional structural modeling of cADPcR, we designed and synthesized cyclic-ADP-4″α-azidoethyl carbocyclic-ribose (N3-cADPcR, 3) to demonstrate that it has a highly potent Ca(2+)-mobilizing activity (EC50 = 24 nM). N3-cADPcR will be a useful precursor for the preparation of biological tools effective to investigate cADPR-mediated signaling pathways. PMID:27391373

  20. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.

  1. Polymerase Chain Reaction for Educational Settings.

    ERIC Educational Resources Information Center

    Garrison, Stephen J.; dePamphillis, Claude

    1994-01-01

    Suggests the incorporation of the Polymerase Chain Reaction (PCR) technique into high school and college biology laboratories. Discusses the following sections: (1) current PCR applications; (2) PCR technique; (3) Manual and Machine PCR; (4) Manual PCR Preparations and Procedure; (5) Materials, Supplies, and Recipes; (6) Primer Selection; and (7)…

  2. A polymerase engineered for bisulfite sequencing

    PubMed Central

    Millar, Doug; Christova, Yonka; Holliger, Philipp

    2015-01-01

    Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains. PMID:26271989

  3. RNA polymerase and the regulation of transcription

    SciTech Connect

    Reznikoff, W.S.; Gross, C.A.; Burgess, R.R.; Record, M.T.; Dahlberg, J.E.; Wickens, M.P.

    1987-01-01

    This book consists of eight sections, each containing several papers. The section titles are: RNA Polymerases; Transcription Initiation - Bacterial; Regulation of Bacterial Transcription Initiation; Stable RNA Synthesis in Eukaryotes: Chromatin Structure; Promoters; Enhancers; and the Global Control of Eukaryotic Transcription; Specific Eukaryotic Transcription Factors; Termination of Transcription; and Short Communications.

  4. Determining Annealing Temperatures for Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Porta, Angela R.; Enners, Edward

    2012-01-01

    The polymerase chain reaction (PCR) is a common technique used in high school and undergraduate science teaching. Students often do not fully comprehend the underlying principles of the technique and how optimization of the protocol affects the outcome and analysis. In this molecular biology laboratory, students learn the steps of PCR with an…

  5. A polymerase engineered for bisulfite sequencing.

    PubMed

    Millar, Doug; Christova, Yonka; Holliger, Philipp

    2015-12-15

    Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.

  6. 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.

  7. Adenosine: A Mediator of the Sleep-Inducing Effects of Prolonged Wakefulness

    PubMed Central

    Porkka-Heiskanen, Tarja; Strecker, Robert E.; Thakkar, Mahesh; Bjørkum, Alvhild A.; Greene, Robert W.; McCarley, Robert W.

    2013-01-01

    Both subjective and electroencephalographic arousal diminish as a function of the duration of prior wakefulness. Data reported here suggest that the major criteria for a neural sleep factor mediating the somnogenic effects of prolonged wakefulness are satisfied by adenosine, a neuromodulator whose extracellular concentration increases with brain metabolism and which, in vitro, inhibits basal forebrain cholinergic neurons. In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness as contrasted with slow wave sleep; exhibited progressive increases during sustained, prolonged wakefulness; and declined slowly during recovery sleep. Furthermore, the sleep-wakefulness profile occurring after prolonged wakefulness was mimicked by increased extracellular adenosine induced by microdialysis perfusion of an adenosine transport inhibitor in the cholinergic basal forebrain but not by perfusion in a control noncholinergic region. PMID:9157887

  8. Passive targeting of ischemic-reperfused myocardium with adenosine-loaded silica nanoparticles

    PubMed Central

    Galagudza, Michael; Korolev, Dmitry; Postnov, Viktor; Naumisheva, Elena; Grigorova, Yulia; Uskov, Ivan; Shlyakhto, Eugene

    2012-01-01

    Pharmacological agents suggested for infarct size limitation have serious side effects when used at cardioprotective doses which hinders their translation into clinical practice. The solution to the problem might be direct delivery of cardioprotective drugs into ischemic-reperfused myocardium. In this study, we explored the potential of silica nanoparticles for passive delivery of adenosine, a prototype cardioprotective agent, into ischemic-reperfused heart tissue. In addition, the biodegradation of silica nanoparticles was studied both in vitro and in vivo. Immobilization of adenosine on the surface of silica nanoparticles resulted in enhancement of adenosine-mediated infarct size limitation in the rat model. Furthermore, the hypotensive effect of adenosine was attenuated after its adsorption on silica nanoparticles. We conclude that silica nanoparticles are biocompatible materials that might potentially be used as carriers for heart-targeted drug delivery. PMID:22619519

  9. Role of CNPase in the oligodendrocytic extracellular 2',3'-cAMP-adenosine pathway.

    PubMed

    Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Bansal, Rashmi; Goebbels, Sandra; Nave, Klaus-Armin; Kochanek, Patrick M; Jackson, Edwin K

    2013-10-01

    Extracellular adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2',3'-cAMP (positional isomer of 3',5'-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2',3'-cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2',3'-cAMP and their respective adenosine monophosphates (2'-AMP and 3'-AMP). Cells were also isolated from mice deficient in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2',3'-cAMP to 2'-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3'-AMP was minimal in both oligodendrocytes and neurons. The production of 2'-AMP from 2',3'-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2'-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3',5'-cAMP-3'-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2',3'-cAMP to 2'-AMP and inhibition of classic ecto-5'-nucleotidase (CD73) with α,β-methylene-adenosine-5'-diphosphate did not attenuate the conversion of 2'-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2',3'-cAMP to 2-AMP in CNS cells. By reducing levels of 2',3'-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. PMID:23922219

  10. Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

    PubMed Central

    Newby, A C

    1980-01-01

    1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells. PMID:6249264

  11. Effect of insulin and glucose on adenosine metabolizing enzymes in human B lymphocytes.

    PubMed

    Kocbuch, Katarzyna; Sakowicz-Burkiewicz, Monika; Grden, Marzena; Szutowicz, Andrzej; Pawelczyk, Tadeusz

    2009-01-01

    In diabetes several aspects of immunity are altered, including the immunomodulatory action of adenosine. Our study was undertaken to investigate the effect of different glucose and insulin concentrations on activities of adenosine metabolizing enzymes in human B lymphocytes line SKW 6.4. The activity of adenosine deaminase in the cytosolic fraction was very low and was not affected by different glucose concentration, but in the membrane fraction of cells cultured with 25 mM glucose it was decreased by about 35% comparing to the activity in cells maintained in 5 mM glucose, irrespective of insulin concentration. The activities of 5'-nucleotidase (5'-NT) and ecto-5'-NT in SKW 6.4 cells depended on insulin concentration, but not on glucose. Cells cultured with 10(-8) M insulin displayed an about 60% lower activity of cytosolic 5'-NT comparing to cells maintained at 10(-11) M insulin. The activity of ecto-5'-NT was decreased by about 70% in cells cultured with 10(-8) M insulin comparing to cells grown in 10(-11) M insulin. Neither insulin nor glucose had an effect on adenosine kinase (AK) activity in SKW 6.4 cells or in human B cells isolated from peripheral blood. The extracellular level of adenosine and inosine during accelerated catabolism of cellular ATP depended on glucose, but not on insulin concentration. Concluding, our study demonstrates that glucose and insulin differentially affect the activities of adenosine metabolizing enzymes in human B lymphocytes, but changes in those activities do not correlate with the adenosine level in cell media during accelerated ATP catabolism, implying that nucleoside transport is the primary factor determining the extracellular level of adenosine.

  12. Role of adenosine in the sympathetic activation produced by isometric exercise in humans.

    PubMed Central

    Costa, F; Biaggioni, I

    1994-01-01

    Isometric exercise increases sympathetic nerve activity and blood pressure. This exercise pressor reflex is partly mediated by metabolic products activating muscle afferents (metaboreceptors). Whereas adenosine is a known inhibitory neuromodulator, there is increasing evidence that it activates afferent nerves. We, therefore, examined the hypothesis that adenosine stimulates muscle afferents and participates in the exercise pressor reflex in healthy volunteers. Intraarterial administration of adenosine into the forearm, during venous occlusion to prevent systemic effects, mimicked the response to exercise, increasing muscle sympathetic nerve activity (MSNA, lower limb microneurography) and mean arterial blood pressure (MABP) at all doses studied (2, 3, and 4 mg). Heart rate increased only with the highest dose. Intrabrachial adenosine (4 mg) increased MSNA by 96 +/- 25% (n = 6, P < 0.01) and MABP by 12 +/- 3 mmHg (P < 0.01). Adenosine produced forearm discomfort, but equivalent painful stimuli (forearm ischemia and cold exposure) increased MSNA significantly less than adenosine. Furthermore, adenosine receptor antagonism with intrabrachial theophylline (1 microgram/ml forearm per min) blocked the increase in MSNA (92 +/- 15% vs. 28 +/- 6%, n = 7, P < 0.01) and MABP (38 +/- 6 vs. 27 +/- 4 mmHg, P = 0.01) produced by isometric handgrip (30% of maximal voluntary contraction) in the infused arm, but not the contralateral arm. Theophylline did not prevent the increase in heart rate produced by handgrip, a response mediated more by central command than muscle afferent activation. We propose that endogenous adenosine contributes to the activation of muscle afferents involved in the exercise pressor reflex in humans. PMID:8163667

  13. Cyclization of the phosphate side chain of adenosine triphosphate: formation of monoadenosine 5'-trimetaphosphate.

    PubMed

    Glonek, T; Kleps, R A; Myers, T C

    1974-07-26

    Monoadenosine 5'-trimetaphosphate has been prepared from adeno-sine 5'-triphosphate by a carbodiimide-mediated condensation. The molecule was characterized by (3l)P nuclear magnetic resonance, and its (31)P spectrum was simulated through the assumption of a three-phosphorus spin system. The molecule is highly reactive and is rapidly converted to adenosine triphosphate upon contact with water. PMID:4834364

  14. Adverse and Protective Influences of Adenosine on the Newborn and Embryo: Implications for Preterm White Matter Injury and Embryo Protection

    PubMed Central

    Rivkees, Scott A.; Wendler, Christopher C.

    2011-01-01

    Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels increase rapidly with tissue hypoxia and inflammation. Adenosine antagonists include the methlyxanthines caffeine and theophylline. The receptors that transduce adenosine action are the A1, A2a, A2b, and A3 adenosine receptors (ARs). In the postnatal period, A1AR activation may contribute to white matter injury in the preterm infant by altering oligodendrocyte (OL) development. In models of perinatal brain injury, caffeine is neuroprotective against periventricular white matter injury (PWMI) and hypoxic-ischemic encephalopathy (HIE). Supporting the notion that blockade of adenosine action is of benefit in the premature infant, caffeine reduces the incidence of broncho-pulmonary dysplasia and cerebral palsy in clinical studies. In comparison with the adverse effects on the postnatal brain, adenosine acts via A1ARs to play an essential role in protecting the embryo from hypoxia. Embryo protective effects are blocked by caffeine, and caffeine intake during early pregnancy increases the risk of miscarriage and fetal growth retardation. Adenosine and adenosine antagonists play important modulatory roles during mammalian development. The protective and deleterious effects of adenosine depend on the time of exposure and target sites of action. PMID:21228731

  15. Ticagrelor Does Not Inhibit Adenosine Transport at Relevant Concentrations: A Randomized Cross-Over Study in Healthy Subjects In Vivo

    PubMed Central

    Rongen, G. A.; van den Broek, P. H. H.; Bilos, A.; Donders, A. R. T.; Gomes, M. E.; Riksen, N. P.

    2015-01-01

    Background and Purpose In patients with myocardial infarction, ticagrelor reduces cardiovascular and sepsis-related mortality, and can cause dyspnea. It is suggested that this is caused by adenosine receptor stimulation, because in preclinical studies, ticagrelor blocks the nucleoside transporter and increases cellular ATP release. We now investigated the effects of ticagrelor on the adenosine system in humans in vivo. Experimental Approach In a double-blinded, placebo-controlled cross-over trial in 14 healthy subjects, we have tested whether ticagrelor (180 mg) affects adenosine- and dipyridamole-induced forearm vasodilation, as surrogates of nucleoside uptake inhibition and adenosine formation, respectively. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was measured. Primary endpoint was adenosine-induced vasodilation. Key Results Ticagrelor did not affect adenosine- or dipyridamole-induced forearm vasodilation. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was not affected by ticagrelor. In vitro, ticagrelor dose-dependently inhibited nucleoside uptake, but only at supra-physiological concentrations. Conclusion and Implications In conclusion, at relevant plasma concentration, ticagrelor does not affect adenosine transport, nor adenosine formation in healthy subjects. Therefore, it is unlikely that this mechanism is a relevant pleiotropic effect of ticagrelor. Trial Registration ClinicalTrials.gov NCT01996735 PMID:26509673

  16. Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors

    PubMed Central

    Paritala, Hanumantharao; Suzuki, Yuta; Carroll, Kate S.

    2015-01-01

    Adenosine 5′-phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of Mycobacterium tuberculosis during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, adenosine 5′-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of N6-substituted adenosine analogues and their 5′-phosphates as well as adenosine 5′-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular docking analysis suggests that the S atom of the most potent inhibitor may establish a favorable interaction with an S atom in the cluster. In summary, this study showcases an improved solid-phase method that expedites the preparation of adenosine and related 5′-phosphate derivatives and presents a unique Fe-S targeting strategy for the development of APR inhibitors. PMID:25710356

  17. Striatal adenosine signaling regulates EAAT2 and astrocytic AQP4 expression and alcohol drinking in mice.

    PubMed

    Lee, Moonnoh R; Ruby, Christina L; Hinton, David J; Choi, Sun; Adams, Chelsea A; Young Kang, Na; Choi, Doo-Sup

    2013-02-01

    Adenosine signaling is implicated in several neuropsychiatric disorders, including alcoholism. Among its diverse functions in the brain, adenosine regulates glutamate release and has an essential role in ethanol sensitivity and preference. However, the molecular mechanisms underlying adenosine-mediated glutamate signaling in neuroglial interaction remain elusive. We have previously shown that mice lacking the ethanol-sensitive adenosine transporter, type 1 equilibrative nucleoside transporter (ENT1), drink more ethanol compared with wild-type mice and have elevated striatal glutamate levels. In addition, ENT1 inhibition or knockdown reduces glutamate transporter expression in cultured astrocytes. Here, we examined how adenosine signaling in astrocytes contributes to ethanol drinking. Inhibition or deletion of ENT1 reduced the expression of type 2 excitatory amino-acid transporter (EAAT2) and the astrocyte-specific water channel, aquaporin 4 (AQP4). EAAT2 and AQP4 colocalization was also reduced in the striatum of ENT1 null mice. Ceftriaxone, an antibiotic compound known to increase EAAT2 expression and function, elevated not only EAAT2 but also AQP4 expression in the striatum. Furthermore, ceftriaxone reduced ethanol drinking, suggesting that ENT1-mediated downregulation of EAAT2 and AQP4 expression contributes to excessive ethanol consumption in our mouse model. Overall, our findings indicate that adenosine signaling regulates EAAT2 and astrocytic AQP4 expressions, which control ethanol drinking in mice.

  18. Label-Free Sensing of Adenosine Based on Force Variations Induced by Molecular Recognition

    PubMed Central

    Li, Jingfeng; Li, Qing; Colombi Ciacchi, Lucio; Wei, Gang

    2015-01-01

    We demonstrate a simple force-based label-free strategy for the highly sensitive sensing of adenosine. An adenosine ssDNA aptamer was bound onto an atomic force microscopy (AFM) probe by covalent modification, and the molecular-interface adsorption force between the aptamer and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). In the presence of adenosine, the molecular recognition between adenosine and the aptamer resulted in the formation of a folded, hairpin-like DNA structure and hence caused a variation of the adsorption force at the graphite/water interface. The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM. The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture. In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated. PMID:25808841

  19. 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

  20. Label-free sensing of adenosine based on force variations induced by molecular recognition.

    PubMed

    Li, Jingfeng; Li, Qing; Ciacchi, Lucio Colombi; Wei, Gang

    2015-03-01

    We demonstrate a simple force-based label-free strategy for the highly sensitive sensing of adenosine. An adenosine ssDNA aptamer was bound onto an atomic force microscopy (AFM) probe by covalent modification, and the molecular-interface adsorption force between the aptamer and a flat graphite surface was measured by single-molecule force spectroscopy (SMFS). In the presence of adenosine, the molecular recognition between adenosine and the aptamer resulted in the formation of a folded, hairpin-like DNA structure and hence caused a variation of the adsorption force at the graphite/water interface. The sensitive force response to molecular recognition provided an adenosine detection limit in the range of 0.1 to 1 nM. The addition of guanosine, cytidine, and uridine had no significant interference with the sensing of adenosine, indicating a strong selectivity of this sensor architecture. In addition, operational parameters that may affect the sensor, such as loading rate and solution ionic strength, were investigated.

  1. Intravenous adenosine (adenoscan) versus exercise in the noninvasive assessment of coronary artery disease by SPECT

    SciTech Connect

    LaManna, M.M.; Mohama, R.; Slavich, I.L. 3d.; Lumia, F.J.; Cha, S.D.; Rambaran, N.; Maranhao, V. )

    1990-11-01

    Fifteen patients at a mean age of 58 underwent adenosine and maximal exercise thallium SPECT imaging. All scans were performed 1 week apart and within 4 weeks of cardiac catheterization. SPECT imaging was performed after the infusion of 140 micrograms/kg/min of adenosine for 6 minutes. Mean heart rate increment during adenosine administration was 67 +/- 3.7 to 77 +/- 4.1. Mean blood pressure was 136 +/- 7.2 to 135 +/- 6.2 systolic and 78 +/- 1.8 to 68 +/- 2.6 diastolic. No adverse hemodynamic effects were observed. There were no changes in PR or QRS in intervals. Five stress ECGs were ischemic. No ST changes were observed with adenosine. Although 68% of the patients had symptoms of flushing, light-headedness, and dizziness during adenosine infusion, symptoms resolved within 1 minute of dosage adjustment or termination of the infusion in all but one patient, who required theophylline. Sensitivity for coronary artery detection was 77% and specificity 100%. Concordance between adenoscans and exercise thallium scintigraphy was high (13/15 = 87%). In two patients, there were minor scintigraphic differences. The authors conclude that adenosine is a sensitive, specific, and safe alternative to exercise testing in patients referred for thallium imaging and may be preferable to dipyridamole.

  2. Ethanol-induced increase in portal blood glow: Role of adenosine

    SciTech Connect

    Orrego, H.; Carmichael, F.J.; Saldivia, V.; Giles, H.G.; Sandrin, S.; Israel, Y. )

    1988-04-01

    The mechanism by which ethanol induces an increase in portal vein blood flow was studied in rats using radiolabeled microspheres. Ethanol by gavage resulted in an increase of 50-70% in portal vein blood flow. The ethanol-induced increase in portal blood flow was suppressed by the adenosine receptor blocker 8-phenyltheophylline. By itself, 8-phenyltheophylline was without effect on cardiac output or portal blood flow. Adenosine infusion resulted in a dose-dependent increase in portal blood flow. This adenosine-induced increase in portal blood flow was inhibited by 8-phenyltheophylline in a dose-dependent manner. Both alcohol and adenosine significantly reduced preportal vascular resistance by 40% and 60%, respectively. These effects were fully suppressed by 8-phenyltheophylline. It is concluded that adenosine is a likely candidate to mediate the ethanol-induced increase in portal vein blood flow. It is suggested that an increase in circulating acetate and liver hypoxia may mediate the effects of alcohol by increasing tissue and interstitial adenosine levels.

  3. Human DNA polymerase. alpha. : Predicted functional domains and relationships with viral DNA polymerases

    SciTech Connect

    Wang, T.S.F.; Wong, S.W.; Korn, D. )

    1989-01-01

    The primary sequence of human DNA polymerase {alpha} deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage {phi}19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DBA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

  4. Adenosine Deaminase Deficiency – More Than Just an Immunodeficiency

    PubMed Central

    Whitmore, Kathryn V.; Gaspar, Hubert B.

    2016-01-01

    Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) that results from mutations in the gene encoding ADA. Affected patients present with clinical and immunological manifestations typical of a SCID. Therapies are currently available that can target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well-understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences. PMID:27579027

  5. Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

    PubMed

    Dokla, Eman M E; Fang, Chun-Sheng; Lai, Po-Ting; Kulp, Samuel K; Serya, Rabah A T; Ismail, Nasser S M; Abouzid, Khaled A M; Chen, Ching-Shih

    2015-11-01

    Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.

  6. Adenosine, type 1 receptors: role in proximal tubule Na+ reabsorption

    PubMed Central

    Welch, William J

    2015-01-01

    Adenosine type 1 receptor (A1-AR) antagonists induce diuresis and natriuresis in experimental animals and humans. Much of this effect is due to inhibition of A1-ARs in the proximal tubule, which is responsible for 60–70% of the reabsorption of filtered Na+ and fluid. Intratubular application of receptor antagonists indicates that A1-AR mediates a portion of Na+ uptake in PT and PT cells, via multiple transport systems, including Na+/H+ exchanger-3 (NHE3), Na+/PO4− co-transporter and Na+-dependent glucose transporter, SGLT. Renal microperfusion and recollection studies have shown that fluid reabsorption is reduced by A1-AR antagonists and is lower in A1-AR KO mice., compared to WT mice. Absolute proximal reabsorption (APR) measured by free-flow micropuncture is equivocal, with studies that show either lower APR or similar APR in A1-AR KO mice, compared to WT mice. Inhibition of A1-ARs lowers elevated blood pressure in models of salt-sensitive hypertension, partially due to their effects in the proximal tubule. PMID:25345761

  7. Adenosine Deaminase Activity in Chronic Lymphocytic Leukemia and Healthy Subjects

    PubMed Central

    Ghaderi, Bayazid; Amini, Sabrieh; Maroofi, Farzad; Jalali, Chiya; Javanmardi, Mitra; Roshani, Daem; Abdi, Mohammad

    2016-01-01

    Background B cell chronic lymphocytic leukemia is one of the most frequent hematologic malignancies in the world. Cellular surface CD markers and serum Beta-2-microglobulin may be used as a prognostic tool in CLL patients. Objectives In the present study we introduce serum adenosine deaminase as a diagnostic marker in CLL. Materials and Methods Blood samples were collected from B-CLL and healthy subjects. White blood cell, red blood cell and platelet count and blood Erythrocyte sedimentation rate was recorded and serum Beta-2-microglobulin, Lactate dehydrogenase and total ADA enzyme activity were determined. Results Serum ADA activity was significantly higher in patients group than that of controls. ADA had a significant and direct correlation with B2M, WBC, LDH and ESR. However, there was not any relation between ADA and the stages of disease. Diagnostic cut-off, sensitivity and specificity of the serum ADA test were 27.97 U/L, 91% and 94%, respectively. Conclusions A higher ADA activity in patients group and its correlation with CLL markers were seen in our study. High diagnostic value of serum ADA in our study suggests that it might be considered as a useful screening tool among the other markers in CLL. PMID:27703646

  8. 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

  9. ADA (adenosine deaminase) gene therapy enters the competition

    SciTech Connect

    Culliton, B.J.

    1990-08-31

    Around the world, some 70 children are members of a select and deadly club. Born with an immune deficiency so severe that they will die of infection unless their immune systems can be repaired, they have captured the attention of would-be gene therapists who believe that a handful of these kids--the 15 or 20 who lack functioning levels of the enzyme adenosine deaminase (ADA)--could be saved by a healthy ADA gene. A team of gene therapists is ready to put the theory to the test. In April 1987, a team of NIH researchers headed by R. Michael Blaese and W. French Anderson came up with the first formal protocol to introduce a healthy ADA gene into an unhealthy human. After 3 years of line-by-line scrutiny by five review committees, they have permission to go ahead. Two or three children will be treated in the next year, and will be infused with T lymphocytes carrying the gene for ADA. If the experiment works, the ADA gene will begin producing normal amounts of ADA. An interesting feature of ADA deficiency, that makes it ideal for initial gene studies, is that the amount of ADA one needs for a healthy immune system is quite variable. Hence, once inside a patient's T cells, the new ADA gene needs only to express the enzyme in moderate amounts. No precise gene regulation is necessary.

  10. Detection of bacteriuria by luciferase assay of adenosine triphosphate.

    PubMed Central

    Thore, A; Anséhn, S; Lundin, A; Bergman, S

    1975-01-01

    A selective method for distinguishing bacterial and nonbacterial adenosine triphosphate (ATP) in clinical bacteriological specimens was studied. The method involved incubation of samples with the detergent Triton X-100 and the ATP-hydrolyzing enzyme apyrase. The incubation selectively destroyed ATP in suspensions of various human cells while not affecting the ATP content in microbial cells. ATP remaining in the sample after incubation was extracted in boiling buffer and assayed by the firefly luciferase assay. Application of the method to 469 clinical urine specimens showed that the ATP level after treatment with Triton/apyrase was correlated to bacterial counts and that the sensitivity of the assay was sufficient for the detection of 10(5) bacteria/ml. The ATP levels per bacterial cell remaining in the urine specimen after treatment with Triton/apyrase were close to values observed in laboratory-grown cultures. The specificity and sensitivity of the luciferase assay for the detection of urinary bacteria and its possible use as a bacteriuria screening method are discussed. PMID:1100645

  11. Sequence specificity of mRNA N6-adenosine methyltransferase.

    PubMed

    Csepany, T; Lin, A; Baldick, C J; Beemon, K

    1990-11-25

    The sequence specificity of chicken mRNA N6-adenosine methyltransferase has been investigated in vivo. Localization of six new N6-methyladenosine sites on Rous sarcoma virus (RSV) virion RNA has confirmed our extended consensus sequence for methylation: RGACU, where R is usually a G (7/12). We have also observed A (2/12) and U (3/12) at the -2 position (relative to m6A at +1) but never a C. At the +3 position, the U was observed 10/12 times; an A and a C were observed once each in weakly methylated sequences. The extent of methylation varied between the different sites up to a maximum of about 90%. To test the significance of this consensus sequence, it was altered by site-specific mutagenesis, and methylation was assayed after transfection of mutated RSV DNA into chicken embryo fibroblasts. We found that changing the G at -1 or the U at +3 to any other residue inhibited methylation. However, inhibition of methylation at all four of the major sites in the RSV src gene did not detectably alter the steady-state levels of the three viral RNA species or viral infectivity. Additional mutants that inactivated the src protein kinase activity produced less virus and exhibited relatively less src mRNA in infected cells. PMID:2173695

  12. Adenosine Deaminase Deficiency - More Than Just an Immunodeficiency.

    PubMed

    Whitmore, Kathryn V; Gaspar, Hubert B

    2016-01-01

    Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) that results from mutations in the gene encoding ADA. Affected patients present with clinical and immunological manifestations typical of a SCID. Therapies are currently available that can target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidence that deficiency of ADA has significant impact on non-immunological organ systems. This review will outline the impact of ADA deficiency on various organ systems, starting with the well-understood immunological abnormalities. We will discuss possible pathogenic mechanisms and also highlight ways in which current treatments could be improved. In doing so, we aim to present ADA deficiency as more than an immunodeficiency and suggest that it should be recognized as a systemic metabolic disorder that affects multiple organ systems. Only by fully understanding ADA deficiency and its manifestations in all organ systems can we aim to deliver therapies that will correct all the clinical consequences. PMID:27579027

  13. Development of Novel Adenosine Monophosphate-Activated Protein Kinase Activators

    PubMed Central

    Guh, Jih-Hwa; Chang, Wei-Ling; Yang, Jian; Lee, Su-Lin; Wei, Shuo; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

    2010-01-01

    In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)γ-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidinedione-based focused compound library to identify novel agents with these dual pharmacological activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homolog of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase, respectively), and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human macrophages were used to screen this compound library, which led to the identification of compound 53 (N-{4-[3-(1-Methylcyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoromethyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore, compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells, indicating that it is not a cell line-specific event. PMID:20170185

  14. Biophysical Mapping of the Adenosine A2A Receptor

    PubMed Central

    2011-01-01

    A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding of small molecule ligands using surface plasmon resonance (SPR) measurement. A matrix of binding data for a set of ligands versus each active site mutation is then generated, providing specific affinity and kinetic information (KD, kon, and koff) of receptor–ligand interactions. This data set, in combination with molecular modeling and docking, is used to map the small molecule binding site for each class of compounds. Taken together, the many constraints provided by these data identify key protein–ligand interactions and allow the shape of the site to be refined to produce a high quality three-dimensional picture of ligand binding, thereby facilitating structure based drug design. Results of biophysical mapping of the adenosine A2A receptor are presented. PMID:21661720

  15. Geometry and cooperativity effects in adenosine-carboxylic acid complexes.

    PubMed

    Schlund, Sebastian; Mladenovic, Milena; Basílio Janke, Eline M; Engels, Bernd; Weisz, Klaus

    2005-11-23

    NMR experiments and theoretical investigations were performed on hydrogen bonded complexes of specifically 1- and 7-15N-labeled adenine nucleosides with carboxylic acids. By employing a freonic solvent of CDClF2 and CDF3, NMR spectra were acquired at temperatures as low as 123 K, where the regime of slow hydrogen bond exchange is reached and several higher-order complexes were found to coexist in solution. Unlike acetic acid, chloroacetic acid forms Watson-Crick complexes with the proton largely displaced from oxygen to the nitrogen acceptor in an ion pairing structure. Calculated geometries and chemical shifts of the proton in the hydrogen bridge favorably agree with experimentally determined values if vibrational averaging and solvent effects are taken into account. The results indicate that binding a second acidic ligand at the adenine Hoogsteen site in a ternary complex weakens the hydrogen bond to the Watson-Crick bound carboxylic acid. However, substituting a second adenine nucleobase for a carboxylic acid in the trimolecular complex leads to cooperative binding at Watson-Crick and Hoogsteen faces of adenosine.

  16. Adenosine Deaminase Inhibition Prevents Clostridium difficile Toxin A-Induced Enteritis in Mice ▿

    PubMed Central

    de Araújo Junqueira, Ana Flávia Torquato; Dias, Adriana Abalen Martins; Vale, Mariana Lima; Spilborghs, Graziela Machado Gruner Turco; Bossa, Aline Siqueira; Lima, Bruno Bezerra; Carvalho, Alex Fiorini; Guerrant, Richard Littleton; Ribeiro, Ronaldo Albuquerque; Brito, Gerly Anne

    2011-01-01

    Toxin A (TxA) is able to induce most of the classical features of Clostridium difficile-associated disease in animal models. The objective of this study was to determine the effect of an inhibitor of adenosine deaminase, EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], on TxA-induced enteritis in C57BL6 mice and on the gene expression of adenosine receptors. EHNA (90 μmol/kg) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) 30 min prior to TxA (50 μg) or PBS injection into the ileal loop. A2A adenosine receptor agonist (ATL313; 5 nM) was injected in the ileal loop immediately before TxA (50 μg) in mice pretreated with EHNA. The animals were euthanized 3 h later. The changes in the tissue were assessed by the evaluation of ileal loop weight/length and secretion volume/length ratios, histological analysis, myeloperoxidase assay (MPO), the local expression of inducible nitric oxide synthase (NOS2), pentraxin 3 (PTX3), NF-κB, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) by immunohistochemistry and/or quantitative reverse transcription-PCR (qRT-PCR). The gene expression profiles of A1, A2A, A2B, and A3 adenosine receptors also were evaluated by qRT-PCR. Adenosine deaminase inhibition, by EHNA, reduced tissue injury, neutrophil infiltration, and the levels of proinflammatory cytokines (TNF-α and IL-1β) as well as the expression of NOS2, NF-κB, and PTX3 in the ileum of mice injected with TxA. ATL313 had no additional effect on EHNA action. TxA increased the gene expression of A1 and A2A adenosine receptors. Our findings show that the inhibition of adenosine deaminase by EHNA can prevent Clostridium difficile TxA-induced damage and inflammation possibly through the A2A adenosine receptor, suggesting that the modulation of adenosine/adenosine deaminase represents an important tool in the management of C. difficile-induced disease. PMID:21115723

  17. Adenosine deaminase inhibition prevents Clostridium difficile toxin A-induced enteritis in mice.

    PubMed

    de Araújo Junqueira, Ana Flávia Torquato; Dias, Adriana Abalen Martins; Vale, Mariana Lima; Spilborghs, Graziela Machado Gruner Turco; Bossa, Aline Siqueira; Lima, Bruno Bezerra; Carvalho, Alex Fiorini; Guerrant, Richard Littleton; Ribeiro, Ronaldo Albuquerque; Brito, Gerly Anne

    2011-02-01

    Toxin A (TxA) is able to induce most of the classical features of Clostridium difficile-associated disease in animal models. The objective of this study was to determine the effect of an inhibitor of adenosine deaminase, EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], on TxA-induced enteritis in C57BL6 mice and on the gene expression of adenosine receptors. EHNA (90 μmol/kg) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) 30 min prior to TxA (50 μg) or PBS injection into the ileal loop. A(2A) adenosine receptor agonist (ATL313; 5 nM) was injected in the ileal loop immediately before TxA (50 μg) in mice pretreated with EHNA. The animals were euthanized 3 h later. The changes in the tissue were assessed by the evaluation of ileal loop weight/length and secretion volume/length ratios, histological analysis, myeloperoxidase assay (MPO), the local expression of inducible nitric oxide synthase (NOS2), pentraxin 3 (PTX3), NF-κB, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) by immunohistochemistry and/or quantitative reverse transcription-PCR (qRT-PCR). The gene expression profiles of A₁, A(2A), A(2B), and A₃ adenosine receptors also were evaluated by qRT-PCR. Adenosine deaminase inhibition, by EHNA, reduced tissue injury, neutrophil infiltration, and the levels of proinflammatory cytokines (TNF-α and IL-1β) as well as the expression of NOS2, NF-κB, and PTX3 in the ileum of mice injected with TxA. ATL313 had no additional effect on EHNA action. TxA increased the gene expression of A₁ and A(2A) adenosine receptors. Our findings show that the inhibition of adenosine deaminase by EHNA can prevent Clostridium difficile TxA-induced damage and inflammation possibly through the A(2A) adenosine receptor, suggesting that the modulation of adenosine/adenosine deaminase represents an important tool in the management of C. difficile-induced disease.

  18. Comonitoring of adenosine and dopamine using the Wireless Instantaneous Neurotransmitter Concentration System: proof of principle

    PubMed Central

    Shon, Young-Min; Chang, Su-Youne; Tye, Susannah J.; Kimble, Christopher J.; Bennet, Kevin E.; Blaha, Charles D.; Lee, Kendall H.

    2010-01-01

    Object The authors of previous studies have demonstrated that local adenosine efflux may contribute to the therapeutic mechanism of action of thalamic deep brain stimulation (DBS) for essential tremor. Real-time monitoring of the neurochemical output of DBS-targeted regions may thus advance functional neurosurgical procedures by identifying candidate neurotransmitters and neuromodulators involved in the physiological effects of DBS. This would in turn permit the development of a method of chemically guided placement of DBS electrodes in vivo. Designed in compliance with FDA-recognized standards for medical electrical device safety, the authors report on the utility of the Wireless Instantaneous Neurotransmitter Concentration System (WINCS) for real-time comonitoring of electrical stimulation–evoked adenosine and dopamine efflux in vivo, utilizing fast-scan cyclic voltammetry (FSCV) at a polyacrylonitrile-based (T-650) carbon fiber microelectrode (CFM). Methods The WINCS was used for FSCV, which consisted of a triangle wave scanned between −0.4 and +1.5 V at a rate of 400 V/second and applied at 10 Hz. All voltages applied to the CFM were with respect to an Ag/AgCl reference electrode. The CFM was constructed by aspirating a single T-650 carbon fiber (r = 2.5 μm) into a glass capillary and pulling to a microscopic tip using a pipette puller. The exposed carbon fiber (the sensing region) extended beyond the glass insulation by ∼ 50 μm. Proof of principle tests included in vitro measurements of adenosine and dopamine, as well as in vivo measurements in urethane-anesthetized rats by monitoring adenosine and dopamine efflux in the dorsomedial caudate putamen evoked by high-frequency electrical stimulation of the ventral tegmental area and substantia nigra. Results The WINCS provided reliable, high-fidelity measurements of adenosine efflux. Peak oxidative currents appeared at +1.5 V and at +1.0 V for adenosine, separate from the peak oxidative current at +0.6 V

  19. Nucleolin Is Required for RNA Polymerase I Transcription In Vivo▿

    PubMed Central

    Rickards, Brenden; Flint, S. J.; Cole, Michael D.; LeRoy, Gary

    2007-01-01

    Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin. PMID:17130237

  20. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477-42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  1. The polymerase chain reaction (PCR): general methods.

    PubMed

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  2. Colony Polymerase Chain Reaction with Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR.

  3. On the role, inactivation and origin of endogenous adenosine at the frog neuromuscular junction.

    PubMed Central

    Ribeiro, J A; Sebastião, A M

    1987-01-01

    1. The effects of adenosine deaminase, inosine, alkylxanthines (8-phenyltheophylline (8-PT), theophylline and isobutylmethylxanthine (IBMX], dipyridamole, alpha, beta-methylene ADP (AOPCP) and ATP analogues (alpha, beta-methylene ATP and beta, gamma-methylene ATP) on evoked end-plate potentials (e.p.p.s) were investigated in innervated sartorius muscles of the frog, in which twitches had been prevented with tubocurarine. The effects of 8-PT and IBMX on the amplitude and quantal content of e.p.p.s were also investigated in innervated sartorius muscles of the frog, in which twitches had been prevented with high-magnesium solutions. 2. Adenosine deaminase reversibly increased the amplitude of e.p.p.s and prevented the reduction caused by exogenously applied adenosine on e.p.p. amplitude. The increase caused by adenosine deaminase was equivalent to the decrease caused by 12 +/- 5.8 microM-adenosine on e.p.p. amplitude. 3. Inosine, the product of adenosine deamination, was virtually devoid of effect on e.p.p.s. 4. The adenosine receptor antagonists at the frog neuromuscular junction, 8-PT and theophylline, increased in a concentration-dependent manner the amplitude of e.p.p.s in the presence of tubocurarine. 8-PT increased the amplitude and quantal content of e.p.p.s in the presence of high magnesium. IBMX, which does not behave as an adenosine receptor antagonist at the frog neuromuscular junction, decreased the amplitude of e.p.p.s in the presence of tubocurarine or high-magnesium solutions. 5. Dipyridamole, an adenosine uptake blocker, decreased the amplitude of e.p.p.s, and in a concentration that did not affect neuromuscular transmission potentiated the depressing effect of adenosine, but not that of 2-chloroadenosine, on the amplitude of e.p.p.s. 6. AOPCP, an inhibitor of 5'-nucleotidase, increased the amplitude of e.p.p.s and markedly attenuated the depressing effect of ATP, but not that of adenosine, on e.p.p. amplitude. 7. The ATP analogue, alpha, beta

  4. Chronic hypoxia enhances adenosine release in rat PC12 cells by altering adenosine metabolism and membrane transport.

    PubMed

    Kobayashi, S; Zimmermann, H; Millhorn, D E

    2000-02-01

    Acute exposure to hypoxia causes a release of adenosine (ADO) that is inversely related to the O2 levels in oxygen-sensitive pheochromocytoma (PC12) cells. In the current study, chronic exposure (48 h) of PC12 cells to moderate hypoxia (5% O2) significantly enhanced the release of ADO during severe, acute hypoxia (1% O2). Investigation into the intra- and extracellular mechanisms underpinning the secretion of ADO in PC12 cells chronically exposed to hypoxia revealed changes in gene expression and activities of several key enzymes associated with ADO production and metabolism, as well as the down-regulation of a nucleoside transporter. Decreases in the enzymatic activities of ADO kinase and ADO deaminase accompanied by an increase in those of cytoplasmic and ecto-5'-nucleotidases bring about an increased capacity to produce intra- and extracellular ADO. This increased potential to generate ADO and decreased capacity to metabolize ADO indicate that PC12 cells shift toward an ADO producer phenotype during hypoxia. The reduced function of the rat equilibrative nucleoside transporter rENT1 also plays a role in controlling extracellular ADO levels. The hypoxia-induced alterations in the ADO metabolic enzymes and the rENT1 transporter seem to increase the extracellular concentration of ADO. The biological significance of this regulation is unclear but is likely to be associated with modulating cellular activity during hypoxia. PMID:10646513

  5. Reticulocyte RNA-Dependent RNA Polymerase

    PubMed Central

    Downey, Kathleen M.; Byrnes, John J.; Jurmark, Bonnie S.; So, Antero G.

    1973-01-01

    A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes. PMID:4519633

  6. Adenosine A2(A) receptor modulates the oxidative stress response of primed polymorphonuclear leukocytes after parabolic flight.

    PubMed

    Kaufmann, Ines; Feuerecker, Matthias; Salam, Alex; Schelling, Gustav; Thiel, Manfred; Choukèr, Alexander

    2011-07-01

    Space flight and gravitational stress can alter innate immune function. Parabolic flights (PFs) as a model for short-term gravitational changes prime the cytotoxic capability of polymorphonuclear leukocytes (PMNs). In view of the emerging role of adenosine in the regulation of innate immune responses, we examined the potency of adenosine to control the release of cytotoxic H(2)O(2) by primed PMNs via the adenosine receptor system. During PFs, microgravity conditions (<10(-2) G) are generated for approximately 22 seconds, followed by a hypergravity (1.8 G) phase resulting in gravitational stress. We studied the ex vivo effects of adenosine on the production of H(2)O(2) by stimulated PMNs and determined adenosine plasma levels and adenosine A2(A) receptor transcripts of leukocytes of PF participants (n = 15). Increasing concentrations of adenosine dose dependently reduced tissue-toxic H(2)O(2) production by PMNs with a half-maximal inhibitory concentration (IC(50)) of 19.5 nM before takeoff and 7.6 nM at 48 hours after PF. This increase in the adenosine-mediated inhibition of PMNs' H(2)O(2) production was completely reversed by addition of the A2(A) receptor antagonist ZM241385. PF induced a nonsignificant elevation in adenosine plasma levels; A2(A) receptor mRNA from leukocytes remained almost unchanged. Adenosine limits the oxidative stress response of PMNs after PFs through an upregulation of the adenosine A2(A) receptor function. This stop signal on inflammation is stronger than that under normal physiologic states and may limit further cytotoxic damage. Pharmacologic manipulation of the adenosine A2(A) receptor pathway could be a potential target for control of unwanted exacerbations of cytotoxic PMN functions.

  7. In vivo adenosine A(2B) receptor desensitization in guinea-pig airway smooth muscle: implications for asthma.

    PubMed

    Breschi, Maria Cristina; Blandizzi, Corrado; Fogli, Stefano; Martinelli, Cinzia; Adinolfi, Barbara; Calderone, Vincenzo; Camici, Marcella; Martinotti, Enrica; Nieri, Paola

    2007-12-01

    This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

  8. Involvement of Peripheral Adenosine A2 Receptors in Adenosine A1 Receptor–Mediated Recovery of Respiratory Motor Function After Upper Cervical Spinal Cord Hemisection

    PubMed Central

    James, Elysia; Nantwi, Kwaku D

    2006-01-01

    Background/Objective: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated through central adenosine A1 receptor antagonism to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although respiration is modulated by central and peripheral mechanisms, putative involvement of peripheral adenosine A2 receptors in functional recovery in our model is untested. The objective of this study was to assess the effects of peripherally located adenosine A2 receptors on recovery of respiratory function after cervical (C2) spinal cord hemisection. Methods: Respiratory activity was electrophysiologically assessed (under standardized recording conditions) in C2-hemisected adult rats with the carotid bodies intact (H-CBI; n =12) or excised (H-CBE; n =12). Animals were administered the adenosine A2 receptor agonist, CGS-21680, followed by the A1 receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX), or administered DPCPX alone. Recovered respiratory activity, characterized as drug-induced activity in the previously quiescent left phrenic nerve of C2-hemisected animals in H-CBI and H-CBE rats, was compared. Recovered respiratory activity was calculated by dividing drug-induced activity in the left phrenic nerve by activity in the right phrenic nerve. Results: Administration of CGS-21680 before DPCPX (n = 6) in H-CBI rats induced a significantly greater recovery (58.5 ± 3.6%) than when DPCPX (42.6 ± 4.6%) was administered (n = 6) alone. In H-CBE rats, prior administration of CGS-21680 (n = 6) did not enhance recovery over that induced by DPCPX (n = 6) alone. Recovery in H-CBE rats amounted to 39.7 ± 3.7% and 38.4 + 4.2%, respectively. Conclusions: Our results suggest that adenosine A2 receptors located in the carotid bodies can enhance the magnitude of adenosine A1 receptor–mediated recovery of respiratory function after C2 hemisection

  9. IL-11 Is Required for A1 Adenosine Receptor–Mediated Protection against Ischemic AKI

    PubMed Central

    Kim, Joo Yun; Kim, Mihwa; Ham, Ahrom; Brown, Kevin M.; Greene, Robert W.; D’Agati, Vivette D.

    2013-01-01

    A1 adenosine receptor activation ameliorates ischemic AKI through the induction of renal proximal tubular sphingosine kinase-1. However, systemic adverse effects may limit A1 adenosine receptor–based therapy for ischemic AKI, indicating a need to identify alternative therapeutic targets within this pathway. Here, we evaluated the function of renal proximal tubular IL-11, a clinically approved hematopoietic cytokine, in A1 adenosine receptor–mediated induction of sphingosine kinase-1 and renal protection. Treatment of human proximal tubule epithelial (HK-2) cells with a selective A1 adenosine receptor agonist, chloro-N(6)-cyclopentyladenosine (CCPA), induced the expression of IL-11 mRNA and protein in an extracellular signal–regulated kinase–dependent manner, and administration of CCPA in mice induced renal synthesis of IL-11. Pretreatment with CCPA protected against renal ischemia-reperfusion injury in wild-type mice, but not in IL-11 receptor–deficient mice. Administration of an IL-11–neutralizing antibody abolished the renal protection provided by CCPA. Similarly, CCPA did not induce renal IL-11 expression or protect against renal ischemia-reperfusion injury in mice lacking the renal proximal tubular A1 adenosine receptor. Finally, treatment with CCPA induced sphingosine kinase-1 in HK-2 cells and wild-type mice, but not in IL-11 receptor–deficient or renal proximal tubule A1 adenosine receptor–deficient mice. Taken together, these results suggest that induction of renal proximal tubule IL-11 is a critical intermediary in A1 adenosine receptor–mediated renal protection that warrants investigation as a novel therapeutic target for the treatment of ischemic AKI. PMID:23813214

  10. Adenosine enhances sweet taste through A2B receptors in the taste bud

    PubMed Central

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D.

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (Type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+ mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (Type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell RT-PCR on isolated vallate taste cells, we show that many Receptor cells express adenosine receptors, Adora2b, while Presynaptic (Type III) and Glial-like (Type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase (ACPP). Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. PMID:22219293

  11. Adenosine enhances sweet taste through A2B receptors in the taste bud.

    PubMed

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

  12. Orexin A attenuates the sleep-promoting effect of adenosine in the lateral hypothalamus of rats.

    PubMed

    Cun, Yanping; Tang, Lin; Yan, Jie; He, Chao; Li, Yang; Hu, Zhian; Xia, Jianxia

    2014-10-01

    Orexin neurons within the lateral hypothalamus play a crucial role in the promotion and maintenance of arousal. Studies have strongly suggested that orexin neurons are an important target in endogenous adenosine-regulated sleep homeostasis. Orexin A induces a robust increase in the firing activity of orexin neurons, while adenosine has an inhibitory effect. Whether the excitatory action of orexins in the lateral hypothalamus actually promotes wakefulness and reverses the sleep-producing effect of adenosine in vivo is less clear. In this study, electroencephalographic and electromyographic recordings were used to investigate the effects of orexin A and adenosine on sleep and wakefulness in rats. We found that microinjection of orexin A into the lateral hypothalamus increased wakefulness with a concomitant reduction of sleep during the first 3 h of post-injection recording, and this was completely blocked by a selective antagonist for orexin receptor 1, SB 334867. The enhancement of wakefulness also occurred after application of the excitatory neurotransmitter glutamate in the first 3 h post-injection. However, in the presence of the NMDA receptor antagonist APV, orexin A did not induce any change of sleep and wakefulness in the first 3 h. Further, exogenous application of adenosine into the lateral hypothalamus induced a marked increase of sleep in the first 3-h post-injection. No significant change in sleep and wakefulness was detected after adenosine application followed by orexin A administration into the same brain area. These findings suggest that the sleep-promoting action of adenosine can be reversed by orexin A applied to the lateral hypothalamus, perhaps by exciting glutamatergic input to orexin neurons via the action of orexin receptor 1.

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

    PubMed Central

    Cusack, N. J.; Planker, M.

    1979-01-01

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

  14. Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

    PubMed Central

    Fuentes, Eduardo; Pereira, Jaime; Mezzano, Diego; Alarcón, Marcelo; Caballero, Julio; Palomo, Iván

    2014-01-01

    Background The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. Objectives The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. Results Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. Conclusion Therefore

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

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

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

  16. Adenosine Phosphates in Germinating Radish (Raphanus sativus L.) Seeds 1

    PubMed Central

    Moreland, Donald E.; Hussey, Griscelda G.; Shriner, Carole R.; Farmer, Fred S.

    1974-01-01

    Changes in concentrations of adenosine phosphates (AMP, ADP, and ATP), oxygen utilization, and fresh weights were measured during the first 48 hours after imbibition of water by quiescent radish seeds (Raphanus sativus L.) at 22.5 C. The changes in ATP concentrations, oxygen utilization, and fresh weights followed a triphasic time course, characterized by a rapid initial increase, which extended from 0 to approximately 1.5 hours, a lag phase from 1.5 to 16 hours, and a sharp linear increase from 16 to 48 hours. In unimbibed seeds, the concentrations of ATP, ADP, and AMP were <0.1, 0.9, and 2.2 nmoles/seed, respectively. After imbibition of water by the quiescent seeds, for 1 hour, the ATP concentration had increased to 2.5, and ADP and AMP concentrations had decreased to 0.3 and 0.1 nmole/seed, respectively. These early changes occurred also in seeds maintained under anaerobic conditions (argon), or when treated with either 5 mm fluoroacetate, or 5 mm iodoacetate. The concentrations of ADP and AMP did not change significantly from 1 to 48 hours. The termination of the lag phase at 16 hours correlated with radicle emergence. Cell division in the radicles was initiated at approximately 28 hours. ATP concentrations in seeds maintained under argon or treated with fluoroacetate remained relatively constant from approximately 2 to 48 hours. In contrast, the ATP concentration of iodoacetate-treated seeds decreased curvilinearly from 4 to 48 hours. Oxidative phosphorylation was estimated to have contributed 15, 20, and 65% of the pool ATP at 1.5, 16, and 48 hours, respectively. PMID:16658928

  17. Equilibrium and kinetic selectivity profiling on the human adenosine receptors.

    PubMed

    Guo, Dong; Dijksteel, Gabrielle S; van Duijl, Tirsa; Heezen, Maxime; Heitman, Laura H; IJzerman, Adriaan P

    2016-04-01

    Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand-receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [(35)S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications.

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

    PubMed

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

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

  19. Volume and ion effects of adenosine on MDCK cells

    SciTech Connect

    Coffey, A.K.; Allen, J.C.; Coutermarsh, B.A.; Mills, J.W.

    1986-03-01

    Adenosine (ADO) receptors, both internal inhibitory (P-site) and external stimulatory (A2) and inhibitory (Al) have been shown to modulate adenylate cyclase levels. Since the authors have previously shown that cAMP modulates ion content and volume of confluent monolayers of MDCK epithelial cells, the authors investigated the effects of ADO on these parameters. Exposure of MDCK cells to 0.1 mM ADO significantly increased cell volumes (2.35 +/- .10 pL/cell vs. control 2.08 +/- .01; p < .001) as determined by /sup 14/C-urea distribution space, and increased Na content (133 +/- 13 vs. 91 +/- 11 nmol/10/sup 6/ cells; p < .0002). Cl/sup -/ content also increased while K did not change. These effects were completely inhibited by treatment with the Na/sup +//H/sup +/ exchange inhibitor amiloride (0.1 mM) and by incubation in Na-free media. Together these results suggest that cell volume increased due to Na/sup +/ entry via the Na/sup +//H/sup +/ exchanger. However, 2-chloroadenosine (2CA), a potent analogue of ADO, caused a significant decrease in cell volume (1.99 +/- .86 vs. 2.21 +/- 0.13 pL/cell; p < .02) and Na (97 +/- 22 vs. 141 +/- 21 nmol/10/sup 6/ cells; p < .005). Measurement of cell cAMP by radioimmunoassay showed an increase in response to 2CA but not to ADO. Since the effects of 2CA mimic those of exogenous cAMP, MDCK cells appear to have a stimulatory (A2) receptor. However, ADO itself did not interact with this receptor and may have produced its effects by binding to a higher affinity, inhibitory receptor.

  20. Purine derivatives as ligands for A3 adenosine receptors.

    PubMed

    Joshi, Bhalchandra V; Jacobson, Kenneth A

    2005-01-01

    Selective agonists and antagonists for A3 adenosine receptors (ARs) are being explored for the treatment of a variety of disorders, including brain and heart ischemic conditions, cancer, and rheumatoid arthritis. This review covers both the structure activity relationships of nucleoside agonist ligands and selected antagonists acting at this receptor and the routes of synthesis. Highly selective agonists have been designed, using both empirical approaches and a semi-rational approach based on molecular modeling. The prototypical A3 agonists IB-MECA 10 and the more receptor-subtype-selective Cl-IB-MECA 11, both of which have affinity in binding to the receptor of approximately 1 nM, have been used widely as pharmacological probes in the elucidation of the physiological role of this receptor. In addition to the exploration of the effects of structural modification of the adenine and ribose moieties on A3AR affinity, the effects of these structural changes on the intrinsic efficacy have also been studied in a systematic fashion. Key structural features determining A3AR interaction include the N6-benzyl group, 2-position substitution such as halo, substitution of ribose (e.g., the (N)-methanocarba ring system, various 2'- and 3'-substitutions and 4'-thio substitution of oxygen). Conformational studies of the ribose moiety and its equivalents indicate that the ring oxygen is not required and the North (N) ring conformation is preferred in binding to the A3AR. Using these observations, a series of ring constrained (N)-methanocarba 5'-uronamide derivatives was recently reported to be highly selective A3AR agonists, the most notable amongst them was MRS3558 113 having a Ki value in binding to the human A3 receptor of 0.3 nM. PMID:16305531

  1. Equilibrium and kinetic selectivity profiling on the human adenosine receptors.

    PubMed

    Guo, Dong; Dijksteel, Gabrielle S; van Duijl, Tirsa; Heezen, Maxime; Heitman, Laura H; IJzerman, Adriaan P

    2016-04-01

    Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand-receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [(35)S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications. PMID:26930564

  2. 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.

  3. 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.

  4. Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases.

    PubMed

    Loh, Ern; Loeb, Lawrence A

    2005-12-01

    DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity. PMID:16230053

  5. Molecular Determinants of CGS21680 Binding to the Human Adenosine A2A Receptor

    PubMed Central

    Edwards, Patricia C.; Leslie, Andrew G. W.

    2015-01-01

    The adenosine A2A receptor (A2AR) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in an active-like conformation bound to the selective agonist 2-[p-(2-carboxyethyl)phenylethyl-amino]-5′-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A2AR structures bound to either CGS21680, 5′-N-ethylcarboxamido adenosine (NECA), UK432097 [6-(2,2-diphenylethylamino)-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-[2-[[1-(2-pyridyl)-4-piperidyl]carbamoylamino]ethyl]purine-2-carboxamide], or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169EL2, His264EL3, Leu2677.32, and Ile2747.39, and the amine group forms a hydrogen bond with the side chain of Ser672.65. Of these residues, only Ile2747.39 is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A2AR bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser672.65 to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser672.65, Glu169EL2, and His264EL3, and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A2AR. PMID:25762024

  6. Adenosine stimulates DNA fragmentation in human thymocytes by Ca(2+)-mediated mechanisms.

    PubMed

    Szondy, Z

    1994-12-15

    Incubation of human thymocytes with an optimum concentration of adenosine and its receptor site agonist, 2-chloroadenosine, induced increases in intracellular cyclic AMP (cAMP) (from a resting 0.6 +/- 0.1 to 4.1 +/- 0.2 pmol/10(7) cells within 5 min) and Ca2+ (from the resting 85 +/- 7 nM to a peak of 210 +/- 25 nM) levels and resulted in internucleosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being 2-p-(carboxyethylphenylethylamino)-5'-carboxyamidoadenosine < 5'-(N-ethylcarboxamide)adenosine < or = cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 microM) selectively depleted a thymocyte subpopulation (15-20% of the total cells) which expressed higher levels of the CD3 molecule and which was found mainly in the CD4+CD8+ double positive immature thymocyte population. DNA fragmentation was prevented by the addition of actinomycin D or cycloheximide to the thymocyte suspension, indicating that this process required both mRNA and protein synthesis. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin and was a result of an adenosine-induced inositol trisphosphate release. Other agents known to elevate intracellular cAMP levels by different mechanisms failed to induce similar DNA fragmentation, but enhanced the effect of adenosine. This suggested a supporting role for cAMP in adenosine-induced DNA fragmentation. Phorbol dibutyrate, a protein kinase. C activator, previously shown to inhibit Ca(2+)-dependent DNA fragmentation and cell killing in human thymocytes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264, 13399-13402], at 60 ng/ml concentration also prevented adenosine-induced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine

  7. Data of expression and purification of recombinant Taq DNA polymerase.

    PubMed

    Fang, Na; Zhong, Niannian; Yang, Yueyang; Guo, Yujian; Ji, Shaoping

    2016-12-01

    Polymerase chain reaction (PCR) technique is widely used in many experimental conditions, and Taq DNA polymerase is critical in PCR process. In this article, the Taq DNA polymerase expression plasmid is reconstructed and the protein product is obtained by rapid purification, ("Rapid purification of high-activity Taq DNA polymerase" (Pluthero, 1993 [1]), "Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli" (Desai and Pfaffle, 1995 [2])). Here we present the production data from protein expression and provide the analysis results of the production from two different vectors. Meanwhile, the purification data is also provided to show the purity of the protein product. PMID:27656666

  8. Adenosine signaling inhibits CIITA-mediated MHC class II transactivation in lung fibroblast cells.

    PubMed

    Fang, Mingming; Xia, Jun; Wu, Xiaoyan; Kong, Hui; Wang, Hong; Xie, Weiping; Xu, Yong

    2013-08-01

    Efficient antigen presentation by major histocompatibility complex (MHC) molecules represents a critical process in adaptive immunity. Class II transactivator (CIITA) is considered the master regulator of MHC class II (MHC II) transcription. Previously, we have shown that CIITA expression is upregulated in smooth muscle cells deficient in A2b adenosine receptor. Here, we report that treatment with the adenosine receptor agonist adenosine-5'N-ethylcarboxamide (NECA) attenuated MHC II transcription in lung fibro-blast cells as a result of CIITA repression. Further analysis revealed that NECA preferentially abrogated CIITA transcription through promoters III and IV. Blockade with a selective A2b receptor antagonist MRS-1754 restored CIITA-dependent MHC II transactivation. Forskolin, an adenylyl cyclase activator, achieved the same effect as NECA. A2b signaling repressed CIITA transcription by altering histone modifications and recruitment of key factors on the CIITA promoters in a STAT1-dependent manner. MRS-1754 blocked the antagonism of transforming growth factor beta (TGF-β) in CIITA induction by interferon gamma (IFN-γ), alluding to a potential dialogue between TGF-β and adenosine signaling pathways. Finally, A2b signaling attenuated STAT1 phosphorylation and stimulated TGF-β synthesis. In conclusion, we have identified an adenosine-A2b receptor-adenylyl cyclase axis that influences CIITA-mediated MHC II transactivation in lung fibroblast cells and as such have provided invaluable insights into the development of novel immune-modulatory strategies.

  9. Content of Adenosine Phosphates and Adenylate Energy Charge in Germinating Ponderosa Pine Seeds

    PubMed Central

    Ching, Te May; Ching, Kim K.

    1972-01-01

    An average of 540 picomoles of total adenosine phosphates was found in the embryo of mature seeds of ponderosa pine (Pinus ponderosa Laws.) and 1140 picomoles in the gametophyte. Adenylate energy charges were 0.44 and 0.26, respectively. After stratification, total adenosine phosphates increased 7-fold and 6-fold in embryo and gametophyte, respectively, and energy charges rose to 0.85 and 0.75. During germination, total adenosine phosphates increased to a 20-fold peak on the 9th day in gametophytic tissue, parallel with the peak of reserve regradation and organellar synthesis, and then decreased. In embryo and seedling, total adenosine phosphates elevated 80-fold with two distinct oscillating increases of AMP and ADP. The oscillating increases occurred before the emergence of radicle and cotyledons during which the highest mitotic index prevailed in all tissues. Energy charges fluctuated between 0.65 at the rapid cell dividing stage to 0.85 at the fully differentiated stage of the seedling, while energy charges remained around 0.75 in the gametophyte. These data indicated that the content of adenosine phosphates of germinating seeds reflects growth, organogenesis, and morphogenesis, and that a compartmentalized energy metabolism must exist in dividing and growing plant cells. PMID:16658212

  10. Lack of tolerance to motor stimulant effects of a selective adenosine A(2A) receptor antagonist.

    PubMed

    Halldner, L; Lozza, G; Lindström, K; Fredholm, B B

    2000-10-20

    It is well known that tolerance develops to the actions of caffeine, which acts as an antagonist on adenosine A(1) and A(2A) receptors. Since selective adenosine A(2A) antagonists have been proposed as adjuncts to 3,4-dihydroxyphenylalanine (L-DOPA) therapy in Parkinson's disease we wanted to examine if tolerance also develops to the selective A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo [1,5-c]pyrimidine (SCH 58261). SCH 58261 (0.1 and 7.5 mg/kg) increased basal locomotion and the motor stimulation afforded by apomorphine. Neither effect was subject to tolerance following long-term treatment with the same doses given intraperitoneally twice daily. There were no adaptive changes in A(1) and A(2A) adenosine receptors or their corresponding messenger RNA or in dopamine D(1) or D(2) receptors. These results demonstrate that the tolerance that develops to caffeine is not secondary to its inhibition of adenosine A(2A) receptors. The results also offer hope that long-term treatment with an adenosine A(2A) receptor antagonist may be possible in man.

  11. Squalenoyl Adenosine Nanoparticles provide Neuroprotection after Stroke and Spinal Cord Injury

    PubMed Central

    Gaudin, Alice; Yemisci, Müge; Eroglu, Hakan; Lepêtre-Mouelhi, Sinda; Turkoglu, Omer Faruk; Dönmez-Demir, Buket; Caban, Seçil; Fevzi Sargon, Mustafa; 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

    2014-01-01

    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, like adenosine, are inefficient upon systemic administration because of their fast metabolisation and rapid clearance from the bloodstream. Here, we show that the conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allow a prolonged circulation of this nucleoside, to provide 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 paper shows, for the first time, that a hydrophilic and rapidly metabolised molecule like adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene. PMID:25420034

  12. Endogenous adenosine modulates stimulation-induced depression at the frog neuromuscular junction.

    PubMed Central

    Meriney, S D; Grinnell, A D

    1991-01-01

    1. Endogenous adenosine, which is produced by enzymatic degradation of ATP released from synaptic vesicles, has been shown to be a potent inhibitor of acetylcholine release from motor nerve terminals. It has been proposed that this auto-inhibition mechanism might contribute significantly to tetanic stimulation-induced depression. 2. Levels of facilitation and depression during a 20 Hz stimulus train differ greatly in different terminals, but are strongly and non-linearly correlated with the terminal's release characteristics (the amount of transmitter released per unit terminal length, or 'release efficacy'). There is a weaker, approximately linear, correlation between depression and release efficacy at 2 Hz stimulation. 3. The effects of both endogenous and exogenously applied adenosine are also highly variable for different nerve terminals. We have shown that much of this variability can be attributed to the release efficacy of each terminal in the case of endogenous effects, and to the size of the nerve terminal in the case of exogenously applied adenosine receptor agonists. 4. When nerve terminals are pooled according to their individual release characteristics, endogenous adenosine can be shown to contribute significantly to stimulation-induced depression of release primarily in terminals that release enough transmitter to generate significant levels of adenosine, but do not release so much transmitter that depletion of releasable quanta is severe. Images Fig. 1 PMID:1688026

  13. The impact of adenosine and A(2B) receptors on glucose homoeostasis.

    PubMed

    Rüsing, D; Müller, C E; Verspohl, E J

    2006-12-01

    Adenosine and adenosine receptor antagonists are involved in glucose homoeostasis. The participating receptors are not known, mainly due to a lack of specific agonists and antagonists, but are reasonable targets for anti-diabetic therapy. The stable, albeit nonselective, adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) (10 microM) reduced glucose-stimulated insulin release from INS-1 cells. This was mimicked by A(1)-(CHA), A(2A)-(CGS-21680) and A(3)-receptor agonists (Cl-IB-MECA). Two newly synthesized A(2B)-receptor antagonists, PSB-53 and PSB-1115, counteracted the inhibitory effect of NECA. These in-vitro effects were mirrored by in-vivo data with respect to CHA, CGS and Cl-IB-MECA. Distinct concentrations of either PSB-53 or PSB-1115 reversed the decrease in plasma insulin induced by NECA. This was not mimicked by a corresponding change in blood glucose. The effect of PSB-1115 was also obvious in diabetic GotoKakizaki rats: plasma insulin was increased whereas blood glucose was unchanged. During most experiments the effects on blood glucose were not impressive probably because of the physiologically necessary homoeostasis. The adenosine levels were not different in normal Wistar rats and in diabetic GotoKakzaki rats. Altogether the A(2B)-receptor antagonists showed an anti-diabetic potential mainly by increasing plasma insulin levels under conditions when the adenosine tonus was elevated in-vivo and increased insulin release in-vitro.

  14. Purification of an Ion-Stimulated Adenosine Triphosphatase from Plant Roots: Association with Plasma Membranes

    PubMed Central

    Hodges, T. K.; Leonard, R. T.; Bracker, C. E.; Keenan, T. W.

    1972-01-01

    A membrane-bound adenosine triphosphatase (EC 3.6.1.3) that requires Mg++ and that is stimulated by monovalent ions has been purified 7- to 8-fold from homogenates of oat (Avena sativa L. Cult. Goodfield) roots by discontinuous sucrose-gradient centrifugation. The enzyme was substrate specific; adenosine triphosphate was hydrolyzed 25 times more rapidly than other nucleoside triphosphates. The membrane fraction containing adenosine triphosphatase was enriched in plasma membranes, which were identified by the presence of a glucan synthetase (EC 2.4.1.12), a high sterol to phospholipid ratio, and by a stain consisting of periodic acid, chromic acid, and phosphotungstic acid that is specific for plant plasma membranes. Oat-root plasma membranes and the associated adenosine triphosphatase were purified on either a 6-layer discontinuous sucrose gradient or on a simplified gradient consisting of only two sucrose layers. These results represent the first demonstration that plant plasma membranes contain an adenosine triphosphatase that is activated by monovalent ions, and this finding further implicates the enzyme in the absorption of inorganic ions by plant roots. Images PMID:16592027

  15. Endogenous adenosine release is involved in the control of heart rate in rats.

    PubMed

    Jammes, Yves; Joulia, Fabrice; Steinberg, Jean Guillaume; Ravailhe, Sylvie; Delpierre, Stéphane; Condo, Jocelyne; Guieu, Regis; Delliaux, Stéphane

    2015-08-01

    Intravenous (i.v.) injections of adenosine exert marked effects on heart rate (HR) and arterial blood pressure (BP), but the role of an endogenous adenosine release by vagal stimulation has not been evaluated. In anaesthetized rats, we examined HR and BP changes induced by 1 min electrical vagal stimulation in the control condition, and then after i.v. injections of (i) atropine, (ii) propranolol, (iii) caffeine, (iv) 8 cyclopentyl-1,3-dipropylxanthine (DPCPX), or (v) dipyridamole to increase the plasma concentration of adenosine (APC). APC was measured by chromatography in the arterial blood before and at the end of vagal stimulation. The decrease in HR in the controls during vagal stimulation was markedly attenuated, but persisted after i.v. injections of atropine and propranolol. When first administered, DPCPX modestly but significantly reduced the HR response to vagal stimulation, but this disappeared after i.v. caffeine administration. Both the HR and BP responses were significantly accentuated after i.v. injection of dipyridamole. Vagal stimulation induced a significant increase in APC, proportional to the magnitude of HR decrease. Our data suggest that the inhibitory effects of electrical vagal stimulations on HR and BP were partly mediated through the activation of A1 and A2 receptors by an endogenous adenosine release. Our experimental data could help to understand the effects of ischemic preconditioning, which are partially mediated by adenosine. PMID:26222197

  16. 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

  17. Obligatory coupling between proton entry and the synthesis of adenosine 5'-triphosphate in Streptococcus lactis.

    PubMed Central

    Maloney, P C

    1977-01-01

    Proton influx was measured after imposition of an electrochemical potential difference for protons (delta muH+) across the cell membrane of the anaerobe, Streptococcus lactis. As delta muH+ was increased, there was an approximately parallel increase in proton entry, until delta muH+ attained 175 to 200 mV. At this point, a new pathway became available for proton entry, allowing an abrupt increase in both the rate and extent of H+ influx. This gated response depended upon the value of delta muH+ itself, and not upon the value of either the membrane potential or the pH gradient. For delta muH+ above 175 to 200 mV, elevated proton entry occurred only in cells having a functional membrane-bound Ca2+-stimulated, Mg2+stimulated adenosine 5'-triphosphatase (EC 3.6.1.3). When present, elevated proton entry coincided with the appearance of net synthesis of adenosine 5'-triphosphate catalyzed by this adenosine 5'-triphosphatase. These observations demonstrate that membrane-bound adenosine 5'-triphosphatase catalyzes an obligatory coupling between the inward movement of protons and synthesis of adenosine 5'-triphosphate. PMID:21165

  18. 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

  19. 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.

  20. Sulfur-Containing 1,3-Dialkylxanthine Derivatives as Selective Antagonists at A1-Adenosine Receptors

    PubMed Central

    Kiriasis, Leonidas; Barone, Suzanne; Bradbury, Barton J.; Kammula, Udai; Campagne, Jean Michel; Secunda, Sherrie; Daly, John W.; Neumeyer, John L.; Pfleiderer, Wolfgang

    2012-01-01

    Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective. PMID:2754711

  1. Poly(ADP-ribose) polymerase inhibition reverses vascular dysfunction after {gamma}-irradiation

    SciTech Connect

    Beller, Carsten J. . E-mail: Carsten.Beller@urz.uni-heidelberg.de; Radovits, Tamas; Seres, Leila; Kosse, Jens; Krempien, Robert; Gross, Marie-Luise; Penzel, Roland; Berger, Irina; Huber, Peter E.; Hagl, Siegfried; Szabo, Csaba; Szabo, Gabor

    2006-08-01

    Purpose: The generation of reactive oxygen species during {gamma}-irradiation may induce DNA damage, leading to activation of the nuclear enzyme poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) culminating in endothelial dysfunction. In the present study, we assessed the effect of PARP inhibition on changes in vascular function after acute and short-term irradiation. Methods and Materials: In the acute experiments, aortic rings were exposed to 20 Gy of {gamma}-irradiation. The aortae were harvested after 1 or 7 days. Two additional groups received the ultrapotent PARP inhibitor, INO-1001, for 1 or 7 days after irradiation. The aortic rings were precontracted by phenylephrine and relaxation to acetylcholine and sodium nitroprusside were studied. Results: The vasoconstrictor response to phenylephrine was significantly lower both acutely and 1 and 7 days after irradiation. Vasorelaxation to acetylcholine and sodium nitroprusside was not impaired acutely after irradiation. One and seven days after irradiation, vasorelaxation to acetylcholine and sodium nitroprusside was significantly enhanced. Treatment with INO-1001 reversed vascular dysfunction after irradiation. Conclusion: Vascular dysfunction was observed 1 and 7 days after irradiation, as evidenced by reduced vasoconstriction, coupled with endothelium-dependent and -independent hyperrelaxation. PARP inhibition restored vascular function and may, therefore, be suitable to reverse vascular dysfunction after irradiation.

  2. Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

    PubMed Central

    Banasik, Marek; Stedeford, Todd; Strosznajder, Robert P; Takehashi, Masanori; Tanaka, Seigo; Ueda, Kunihiro

    2011-01-01

    Carbon tetrachloride (CCl4) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of (β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl4-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation,and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl4 with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl4 exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl4 and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl) ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl4is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl4. PMID:21395487

  3. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.

    PubMed

    Brownstein, M J; Carpten, J D; Smith, J R

    1996-06-01

    Taq DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called "PIG-tailing") should facilitate accurate genotyping and efficient T/A cloning.

  4. Mutational clusters generated by non-processive polymerases: A case study using DNA polymerase betain vitro.

    PubMed

    García-Villada, Libertad; Drake, John W

    2010-08-01

    Available DNA mutational spectra reveal that the number of mutants with multiple mutations ("multiples") is usually greater than expected from a random distribution of mutations among mutants. These overloads imply the occurrence of non-random clusters of mutations, probably generated during episodes of low-fidelity DNA synthesis. Excess multiples have been reported not only for viruses, bacteria, and eukaryotic cells but also for the DNA polymerases of phages T4 and RB69 in vitro. In the simplest case of a purified polymerase, non-random clusters may be generated by a subfraction of phenotypic variants able to introduce more errors per cycle of DNA synthesis than the normal enzyme. According to this hypothesis, excess multiples are not expected with non-processive polymerases even if they harbor rare mutator variants. DNA polymerase beta (Pol beta) is a mammalian DNA-repair polymerase with very low processivity. Although several Pol beta mutational spectra have been described, there is conflicting evidence on whether or not excess multiples occur, with spectra based on the HSV-tk system tending to show excess multiples. Excess multiples generated by Pol beta or any of its mutants might imply that the excesses of multiples observed in numerous other systems, especially those with processive polymerases, could be artifactual. Here, the distributions of mutations generated by native and recombinant rat Pol beta and by the Pol beta(Y265C) mutator were analyzed in the M13mp2 lacZalpha system. Our results present no evidence for a significant excess of multiples over the expected numbers with any of the Pol beta enzymes tested in this system. The reported excess of Pol beta-generated multiples in the HSV-tk system may reflect a reduced efficiency of detection of base substitutions that cause weak phenotypes, which in turn may artifactually increase the frequency of multiples. PMID:20627824

  5. An ultraviolet-inducible adenosine-adenosine cross-link reflects the catalytic structure of the Tetrahymena ribozyme

    SciTech Connect

    Downs, W.D.; Cech, T.R. )

    1990-06-12

    When a shortened enzymatic version of the Tetrahymena self-splicing intervening sequence (IVS) RNA is placed under catalytic conditions and irradiated at 254 nm, a covalent cross-link forms with high efficiency. The position of the cross-link was mapped by using three independent methods: RNase H digestion, primer extension with reverse transcriptase, and partial hydrolysis of end-labeled RNA. The cross-link is chemically unusual in that it joins two adenosines, A57 and A95. Formation of this cross-link depends upon the identity and concentration of divalent cations present and upon heat-cool renaturation of the IVS in a manner that parallels conditions required for optimal catalytic activity. Furthermore, cross-linking requires the presence of sequences within the core structure, which is conserved among group I intervening sequences and necessary for catalytic activity. Together these correlations suggest that a common folded structure permits cross-linking and catalytic activity. The core can form this structure independent of the presence of P1 and elements at the 3' end of the IVS. The cross-linked RNA loses catalytic activity under destabilizing conditions, presumably due to disruption of the folded structure by the cross-link. One of the nucleotides participating in this cross-link is highly conserved (86%) within the secondary structure of group I intervening sequences. We conclude that A57 and A95 are precisely aligned in a catalytically active conformation of the RNA. A model is presented for the tertiary arrangement in the vicinity of the cross-link.

  6. DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria.

    PubMed

    Fioravanti, Antonella; Fumeaux, Coralie; Mohapatra, Saswat S; Bompard, Coralie; Brilli, Matteo; Frandi, Antonio; Castric, Vincent; Villeret, Vincent; Viollier, Patrick H; Biondi, Emanuele G

    2013-05-01

    Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

  7. A 30-year-old female Behçet's disease patient with recurrent pleural and pericardial effusion and elevated adenosine deaminase levels: case report.

    PubMed

    Choi, Joon Young; Kim, Sung-Hwan; Kwok, Seung-Ki; Jung, Jung Im; Lee, Kyo-Young; Kim, Tae-Jung; Kang, Ji Young

    2016-07-01

    Behçet's disease is a systemic disease which may involve various organs. We describe a case of a patient diagnosed as pleuropericardial involvement of Behçet's disease. A 30-year-old woman visited our clinic presented with left pleuritic chest pain for s days. She had been diagnosed as Behçet's disease and admitted to our clinic due to pericardial and pleural effusion repeatedly in past two years. In the previous studies, effusion analysis revealed to be lympho-dominant exudate with high adenosine deaminase level. Acid-fast bacilli (AFB) culture and polymerase chain reaction (PCR) for mycobacterial tuberculosis (M.TB) were negative in the pericardial tissue, and pathologic finding showed mild endothelitis with micro-thrombi formation in the lumen. The patient had been treated with antituberculous medication for a year. In the current admission, chest computed tomography (CT) again showed left pleural effusion without other significant lesion. Pleural fluid analysis was similar with the previous study. Video-assisted thoracoscopic pleural biopsy was performed to obtain the definite diagnosis. Pathology confirmed the diagnosis as pleuropericardial involvement of Behçet's disease, and we treated the patient with oral steroid in the out-patient department. Pleuropericardial involvement of Behçet's disease may mimic TB pleurisy or pericarditis due to high adenosine deaminase (ADA) level in effusion analysis. Clinicians should keep in mind that Behçet's disease may manifest as pleural or pericardial effusion, and pathologic confirmation could be helpful for the definite diagnosis. PMID:27499994

  8. A 30-year-old female Behçet’s disease patient with recurrent pleural and pericardial effusion and elevated adenosine deaminase levels: case report

    PubMed Central

    Choi, Joon Young; Kim, Sung-Hwan; Kwok, Seung-Ki; Jung, Jung Im; Lee, Kyo-Young; Kim, Tae-Jung

    2016-01-01

    Behçet’s disease is a systemic disease which may involve various organs. We describe a case of a patient diagnosed as pleuropericardial involvement of Behçet’s disease. A 30-year-old woman visited our clinic presented with left pleuritic chest pain for s days. She had been diagnosed as Behçet’s disease and admitted to our clinic due to pericardial and pleural effusion repeatedly in past two years. In the previous studies, effusion analysis revealed to be lympho-dominant exudate with high adenosine deaminase level. Acid-fast bacilli (AFB) culture and polymerase chain reaction (PCR) for mycobacterial tuberculosis (M.TB) were negative in the pericardial tissue, and pathologic finding showed mild endothelitis with micro-thrombi formation in the lumen. The patient had been treated with antituberculous medication for a year. In the current admission, chest computed tomography (CT) again showed left pleural effusion without other significant lesion. Pleural fluid analysis was similar with the previous study. Video-assisted thoracoscopic pleural biopsy was performed to obtain the definite diagnosis. Pathology confirmed the diagnosis as pleuropericardial involvement of Behçet’s disease, and we treated the patient with oral steroid in the out-patient department. Pleuropericardial involvement of Behçet’s disease may mimic TB pleurisy or pericarditis due to high adenosine deaminase (ADA) level in effusion analysis. Clinicians should keep in mind that Behçet’s disease may manifest as pleural or pericardial effusion, and pathologic confirmation could be helpful for the definite diagnosis. PMID:27499994

  9. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats

    PubMed Central

    Iglesias, Inmaculada; Albasanz, Jose Luis

    2014-01-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine. PMID:25538864

  10. Predictors and Diagnostic Significance of the Adenosine Related Side Effects on Myocardial Perfusion SPECT/CT Imaging

    PubMed Central

    Yıldırım Poyraz, Nilüfer; Özdemir, Elif; Poyraz, Barış Mustafa; Kandemir, Zuhal; Keskin, Mutlay; Türkölmez, Şeyda

    2014-01-01

    Objective: The aim of this study was to investigate the relationship between patient characteristics and adenosine-related side-effects during stress myocard perfusion imaging (MPI). The effect of presence of adenosine-related side-effects on the diagnostic value of MPI with integrated SPECT/CT system for coronary artery disease (CAD), was also assessed in this study. Methods: Total of 281 patients (109 M, 172 F; mean age:62.6±10) who underwent standard adenosine stress protocol for MPI, were included in this study. All symptoms during adenosine infusion were scored according to the severity and duration. For the estimation of diagnostic value of adenosine MPI with integrated SPECT/CT system, coronary angiography (CAG) or clinical follow-up were used as gold standard. Results: Total of 173 patients (61.6%) experienced adenosine-related side-effects (group 1); flushing, dyspnea, and chest pain were the most common. Other 108 patients completed pharmacologic stress (PS) test without any side-effects (group 2). Test tolerability were similar in the patients with cardiovascular or airway disease to others, however dyspnea were observed significantly more common in patients with mild airway disease. Body mass index (BMI) ≥30 kg/m2 and age ≤45 years were independent predictors of side-effects. The diagnostic value of MPI was similar in both groups. Sensitivity of adenosine MPI SPECT/CT was calculated to be 86%, specificity was 94% and diagnostic accuracy was 92% for diagnosis of CAD. Conclusion: Adenosine MPI is a feasible and well tolerated method in patients who are not suitable for exercise stress test as well as patients with cardiopulmonary disease. However age ≤45 years and BMI ≥30 kg/m2 are the positive predictors of adenosine-related side-effects, the diagnostic value of adenosine MPI SPECT/CT is not affected by the presence of adenosine related side-effects. PMID:25541932

  11. Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

    PubMed

    Silva-Ramos, M; Silva, I; Faria, M; Magalhães-Cardoso, M T; Correia, J; Ferreirinha, F; Correia-de-Sá, P

    2015-12-01

    This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients.

  12. The role of adenosine receptors in regulating production of tumour necrosis factor-α and chemokines by human lung macrophages

    PubMed Central

    Buenestado, A; Delyle, S Grassin; Arnould, I; Besnard, F; Naline, E; Blouquit-Laye, S; Chapelier, A; Bellamy, JF; Devillier, P

    2010-01-01

    Background and purpose: Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A1, A2A, A2B and A3). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-α and chemokines. Experimental approach: Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. Key results: LPS increased (about 400-fold) mRNA for A2A adenosine receptors, decreased mRNA for A1 and A2B, but had no effect on A3 adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-α production concentration dependently, whereas the A1 receptor agonist, CCPA, and the A3 receptor agonist, AB-MECA, inhibited TNF-α production only at concentrations affecting A2A receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-α and chemokine production by the selective A2A adenosine receptor antagonist ZM 241385, but not the A2B receptor antagonist, MRS 1754, or the A3 receptor antagonist, MRS 1220, indicated involvement of A2A receptors. Conclusions and implications: LPS up-regulated A2A adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-α and of a subset of chemokines in human lung macrophages. PMID:20136829

  13. RNA polymerase molecular beacon as tool for studies of RNA polymerase-promoter interactions.

    PubMed

    Mekler, Vladimir; Severinov, Konstantin

    2015-09-15

    The molecular details of formation of transcription initiation complex upon the interaction of bacterial RNA polymerase (RNAP) with promoters are not completely understood. One way to address this problem is to understand how RNAP interacts with different parts of promoter DNA. A recently developed fluorometric RNAP molecular beacon assay allows one to monitor the RNAP interactions with various unlabeled DNA probes and quantitatively characterize partial RNAP-promoter interactions. This paper focuses on methodological aspects of application of this powerful assay to study the mechanism of transcription initiation complex formation by Escherichia coli RNA polymerase σ(70) holoenzyme and its regulation by bacterial and phage encoded factors.

  14. RNA polymerase molecular beacon as tool for studies of RNA polymerase - promoter interactions

    PubMed Central

    Mekler, Vladimir; Severinov, Konstantin

    2015-01-01

    The molecular details of formation of transcription initiation complex upon the interaction of bacterial RNA polymerase (RNAP) with promoters are not completely understood. One way to address this problem is to understand how RNAP interacts with different parts of promoter DNA. A recently developed fluorometric RNAP molecular beacon assay allows one to monitor the RNAP interactions with various unlabeled DNA probes and quantitatively characterize partial RNAP-promoter interactions. This paper focuses on methodological aspects of application of this powerful assay to study the mechanism of transcription initiation complex formation by Escherichia coli RNA polymerase σ70 holoenzyme and its regulation by bacterial and phage encoded factors. PMID:25956222

  15. ppGpp: magic beyond RNA polymerase.

    PubMed

    Dalebroux, Zachary D; Swanson, Michele S

    2012-02-16

    During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

  16. 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

    2014-08-01

    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.

  17. Role of adenosine in the antiepileptic effects of deep brain stimulation

    PubMed Central

    Miranda, Maisa F.; Hamani, Clement; de Almeida, Antônio-Carlos G.; Amorim, Beatriz O.; Macedo, Carlos E.; Fernandes, Maria José S.; Nobrega, José N.; Aarão, Mayra C.; Madureira, Ana Paula; Rodrigues, Antônio M.; Andersen, Monica L.; Tufik, Sergio; Mello, Luiz E.; Covolan, Luciene

    2014-01-01

    Despite the effectiveness of anterior thalamic nucleus (AN) deep brain stimulation (DBS) for the treatment of epilepsy, mechanisms responsible for the antiepileptic effects of this therapy remain elusive. As adenosine modulates neuronal excitability and seizure activity in animal models, we hypothesized that this nucleoside could be one of the substrates involved in the effects of AN DBS. We applied 5 days of stimulation to rats rendered chronically epileptic by pilocarpine injections and recorded epileptiform activity in hippocampal slices. We found that slices from animals given DBS had reduced hippocampal excitability and were less susceptible to develop ictal activity. In live animals, AN DBS significantly increased adenosine levels in the hippocampus as measured by microdialysis. The reduced excitability of DBS in vitro was completely abolished in animals pre-treated with A1 receptor antagonists and was strongly potentiated by A1 receptor agonists. We conclude that some of the antiepileptic effects of DBS may be mediated by adenosine. PMID:25324724

  18. Autoradiographic localization of adenosine receptors in rat brain using (/sup 3/H)cyclohexyladenosine

    SciTech Connect

    Goodman, R.R.; Synder, S.H.

    1982-09-01

    Adenosine (A1) receptor binding sites have been localized in rat brain by an in vitro light microscopic autoradiographic method. The binding of (/sup 3/H)N6-cyclohexyladenosine to slide-mounted rat brain tissue sections has the characteristics of A1 receptors. It is saturable with high affinity and has appropriate pharmacology and stereospecificity. The highest densities of adenosine receptors occur in the molecular layer of the cerebellum, the molecular and polymorphic layers of the hippocampus and dentate gyrus, the medial geniculate body, certain thalamic nuclei, and the lateral septum. High densities also are observed in certain layers of the cerebral cortex, the piriform cortex, the caudate-putamen, the nucleus accumbens, and the granule cell layer of the cerebellum. Most white matter areas, as well as certain gray matter areas, such as the hypothalamus, have negligible receptor concentrations. These localizations suggest possible central nervous system sites of action of adenosine.

  19. Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex

    SciTech Connect

    Florio, C.; Rosati, A.M.; Traversa, U.; Vertua, R. )

    1991-01-01

    In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6-cyclohexyl-({sup 3}H)adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks. Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.

  20. Activation of two sites by adenosine receptor agonists to cause relaxation in rat isolated mesenteric artery

    PubMed Central

    Prentice, D J; Payne, S L; Hourani, S M O

    1997-01-01

    In this study we have characterized the receptor(s) in the rat mesenteric artery mediating relaxant responses to adenosine and a number of adenosine analogues, N6 -R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), N6-(3-iodo-benzyl)-adenosine-5′-N-methyluronamide (IB-MECA) and 5′-N-ethylcarboxamidoadenosine (NECA), by use of the non-selective antagonist 8-sulphophenyltheophylline (8-SPT) and the A2A selective ligands 2-[p-(2-carbonylethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]-triazin-5-ylamino]ethyl) phenol (ZM 241385). We have also studied the effects of endothelial removal and uptake inhibition by nitrobenzylthioinosine (NBTI) and the effects of the A3 receptor antagonist 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BWA1433).Adenosine, NECA, CPA and R-PIA all elicited relaxant responses in tissues precontracted with phenylephrine (1 μM) with the following potency order: NECA>R-PIA>adenosine=CPA. However, E/[A] curves to NECA were biphasic. CGS 21680 was inactive at concentrations up to 30 μM and IB-MECA elicited relaxant responses which were resistant to blockade by 8-SPT and BWA1433 (100 μM).Removal of the endothelium produced a small but significant decrease in the asymptote of the high potency phase of E/[A] curves to NECA with no change in p[A]50. E/[A] curves to adenosine were not altered by removal of the endothelium. However, there were small rightward shifts of E/[A] curves to CPA and R-PIA in the absence of endothelium.Inhibition of uptake by NBTI (1 μM) had no effect on E/[A] curves to NECA, CPA or R-PIA, but E/[A] curves to adenosine were significantly left-shifted in the presence of NBTI.8-SPT (10–100 μM) caused significant rightward shifts of the high potency phase of the E/[A] curves to NECA (pA2=5.63±0.26). The second phase of the concentration-response curve to NECA appeared to be resistant to blockade by 8-SPT, as were E

  1. The Three Possible 2-(Pyrenylethynyl) Adenosines: Rotameric Energy Barriers Govern the Photodynamics of These Structural Isomers.

    PubMed

    Reuss, Andreas J; Grünewald, Christian; Braun, Markus; Engels, Joachim W; Wachtveitl, Josef

    2016-05-01

    This article presents a comprehensive study of the photophysics of 2-(2-pyrenylethynyl) adenosine and 2-(4-pyrenylethynyl) adenosine, which are structural isomers of the well-established fluorescent RNA label 2-(1-pyrenylethynyl) adenosine. We performed steady-state and ultrafast transient absorption spectroscopy studies along with time-resolved fluorescence emission experiments in different solvents to work out the interplay of locally excited and charge-transfer states. We found the ultrafast photodynamics to be crucial for the fluorescence decay behavior, which extends up to tens of nanoseconds and is partially multi-exponential. These features in the ultrafast dynamics are indicative of the rotational energy barriers in the first excited state.

  2. Computational study of the molecular mechanisms of caffeine action: Caffeine complexes with adenosine receptors

    NASA Astrophysics Data System (ADS)

    Poltev, V. I.; Rodríguez, E.; Grokhlina, T. I.; Deriabina, A.; Gonzalez, E.

    To understand the molecular basis of the principal biological action of the caffeine (CAF), the molecular mechanics calculations of possible complexes between CAF and the fragments of human A1 adenosine receptor were performed. The fragments were selected after considerations of the CAF molecular structure and its possible interactions, as well as after an analysis of the extensive bibliography on the structure, biological role, site-directed mutagenesis, and the modeling of the adenosine receptors. The minimum energy configurations of these complexes were obtained using two different computer programs with different force fields. The most favorable configurations correspond to the formation of two hydrogen bonds between the CAF molecule and hydrophilic amino acid residues of the fragments of transmembrane domains of the receptor. These configurations are supposed to contribute to CAF blocking of the adenosine receptors. They will be used later for the construction of model CAF complexes with two transmembrane domains simultaneously.

  3. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  4. Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes.

    PubMed

    Ataie, G; Moosavi-Movahedi, A A; Saboury, A A; Hakimelahi, G H; Hwu, J R; Tsay, S C

    2000-03-16

    Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18000, 22000 and 21900 kJ mol(-1) respectively.

  5. Autoradiographic localization of adenosine uptake sites in rat brain using (/sup 3/H)nitrobenzylthioinosine

    SciTech Connect

    Bisserbe, J.C.; Patel, J.; Marangos, P.J.

    1985-02-01

    The adenosine uptake site has been localized in rat brain by an in vitro light microscopic autoradiographic method, using (/sup 3/H)nitrobenzylthioinosine ((/sup 3/H)NBI) as the probe. The binding characteristics of (/sup 3/H)NBI on slide-mounted sections are comparable to those seen in studies performed on brain homogenates. A very high density of uptake sites occurs in the nucleus tractus solitarius, in the superficial layer of the superior colliculus, in several thalamic nuclei, and also in geniculate body nuclei. A high density of sites are also observed in the nucleus accumbens, the caudate putamen, the dorsal tegmentum area, the substantia nigra, and the central gray. The localization of the adenosine uptake site in brain may provide information on the functional activity of the site and suggests the involvement of the adenosine system in the central regulation of cardiovascular function.

  6. Caffeine's Attenuation of Cocaine-Induced Dopamine Release by Inhibition of Adenosine

    PubMed Central

    Malave, Lauren B.

    2014-01-01

    Background: It is well known that the reinforcing properties of cocaine addiction are caused by the sharp increase of dopamine (DA) in the reward areas of the brain. However, other mechanisms have been speculated to contribute to the increase. Adenosine is one system that is associated with the sleep-wake cycle and is most important in regulating neuronal activity. Thus, more and more evidence is pointing to its involvement in regulating DA release. The current study set out to examine the role of adenosine in cocaine-induced DA release. Methods: Increasing doses of cocaine, caffeine, and their combination, as well as, 8-cyclopentyltheophylline (CPT), an adenosine A1 antagonist (alone and in combination with cocaine) were used to denote a response curve. A novel biosensor, the BRODERICK PROBE® was implanted in the nucleus accumbens to image the drug-induced surge of DA release in vivo, in the freely moving animal in real time. Results: Combinations of cocaine and caffeine were observed to block the increased release of DA moderately after administration of the low dose (2.5 mg/kg cocaine and 12.5 mg/kg caffeine) and dramatically after administration of the high dose (10 mg/kg cocaine and 50 mg/kg caffeine), suggesting neuroprotection. Similarly, CPT and cocaine showed a decreased DA surge when administered in combination. Thus, the low and high dose of a nonselective adenosine antagonist, caffeine, and a moderate dose of a selective adenosine antagonist, CPT, protected against the cocaine-induced DA release. Conclusions: These results show a significant interaction between adenosine and DA release and suggest therapeutic options for cocaine addiction and disorders associated with DA dysfunction. PMID:25054079

  7. Caffeine's Attenuation of Cocaine-Induced Dopamine Release by Inhibition of Adenosine.

    PubMed

    Malave, Lauren B; Broderick, Patricia A

    2014-06-01

    Background: It is well known that the reinforcing properties of cocaine addiction are caused by the sharp increase of dopamine (DA) in the reward areas of the brain. However, other mechanisms have been speculated to contribute to the increase. Adenosine is one system that is associated with the sleep-wake cycle and is most important in regulating neuronal activity. Thus, more and more evidence is pointing to its involvement in regulating DA release. The current study set out to examine the role of adenosine in cocaine-induced DA release. Methods: Increasing doses of cocaine, caffeine, and their combination, as well as, 8-cyclopentyltheophylline (CPT), an adenosine A1 antagonist (alone and in combination with cocaine) were used to denote a response curve. A novel biosensor, the BRODERICK PROBE(®) was implanted in the nucleus accumbens to image the drug-induced surge of DA release in vivo, in the freely moving animal in real time. Results: Combinations of cocaine and caffeine were observed to block the increased release of DA moderately after administration of the low dose (2.5 mg/kg cocaine and 12.5 mg/kg caffeine) and dramatically after administration of the high dose (10 mg/kg cocaine and 50 mg/kg caffeine), suggesting neuroprotection. Similarly, CPT and cocaine showed a decreased DA surge when administered in combination. Thus, the low and high dose of a nonselective adenosine antagonist, caffeine, and a moderate dose of a selective adenosine antagonist, CPT, protected against the cocaine-induced DA release. Conclusions: These results show a significant interaction between adenosine and DA release and suggest therapeutic options for cocaine addiction and disorders associated with DA dysfunction. PMID:25054079

  8. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    PubMed Central

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  9. Myocardial blood flow and adenosine A2A receptor density in endurance athletes and untrained men

    PubMed Central

    Heinonen, Ilkka; Nesterov, Sergey V; Liukko, Kaisa; Kemppainen, Jukka; Någren, Kjell; Luotolahti, Matti; Virsu, Pauliina; Oikonen, Vesa; Nuutila, Pirjo; Kujala, Urho M; Kainulainen, Heikki; Boushel, Robert; Knuuti, Juhani; Kalliokoski, Kari K

    2008-01-01

    Previous human studies have shown divergent results concerning the effects of exercise training on myocardial blood flow (MBF) at rest or during adenosine-induced hyperaemia in humans. We studied whether these responses are related to alterations in adenosine A2A receptor (A2AR) density in the left-ventricular (LV) myocardium, size and work output of the athlete's heart, or to fitness level. MBF at baseline and during intravenous adenosine infusion, and A2AR density at baseline were measured using positron emission tomography, and by a novel A2AR tracer in 10 healthy male endurance athletes (ET) and 10 healthy untrained (UT) men. Structural LV parameters were measured with echocardiography. LV mass index was 71% higher in ET than UT (193 ± 18 g m−2versus 114 ± 13 g m−2, respectively). MBF per gram of tissue was significantly lower in the ET than UT at baseline, but this was only partly explained by reduced LV work load since MBF corrected for LV work was higher in ET than UT, as well as total MBF. The MBF during adenosine-induced hyperaemia was reduced in ET compared to UT, and the fitter the athlete was, the lower was adenosine-induced MBF. A2AR density was not different between the groups and was not coupled to resting or adenosine-mediated MBF. The novel findings of the present study show that the adaptations in the heart of highly trained endurance athletes lead to relative myocardial ‘overperfusion’ at rest. On the other hand hyperaemic perfusion is reduced, but is not explained by A2AR density. PMID:18772204

  10. Adenosine transport systems on dissociated brain cells from mouse, guinea-pig, and rat

    SciTech Connect

    Johnston, M.E.; Geiger, J.D. )

    1990-09-01

    The kinetics and sodium dependence of adenosine transport were determined using an inhibitor-stop method on dissociated cell body preparations obtained from mouse, guinea-pig and rat brain. Transport affinity (KT) values for the high affinity adenosine transport systems KT(H) were significantly different between these three species; mean +/- SEM values were 0.34 +/- 0.1 in mouse, 0.9 +/- 0.2 in rat, and 1.5 +/- 0.5 microM in guinea-pig. The KT values for the low affinity transport system KT(L) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate (3H)adenosine (Vmax) than did mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the KT(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between KT(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse, guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.

  11. Solving the RNA polymerase I structural puzzle

    SciTech Connect

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  12. Targeting DNA Polymerase β for Therapeutic Intervention

    PubMed Central

    Goellner, Eva M.; Svilar, David; Almeida, Karen H.; Sobol, Robert W.

    2014-01-01

    DNA damage plays a causal role in numerous disease processes. Hence, it is suggested that DNA repair proteins, which maintain the integrity of the nuclear and mitochondrial genomes, play a critical role in reducing the onset of multiple diseases, including cancer, diabetes and neurodegeneration. As the primary DNA polymerase involved in base excision repair, DNA polymerase β (Polβ) has been implicated in multiple cellular processes, including genome maintenance and telomere processing and is suggested to play a role in oncogenic transformation, cell viability following stress and the cellular response to radiation, chemotherapy and environmental genotoxicants. Therefore, Polβ inhibitors may prove to be effective in cancer treatment. However, Polβ has a complex and highly regulated role in DNA metabolism. This complicates the development of effective Polβ-specific inhibitors useful for improving chemotherapy and radiation response without impacting normal cellular function. With multiple enzymatic activities, numerous binding partners and complex modes of regulation from post-translational modifications, there are many opportunities for Polβ inhibition that have yet to be resolved. To shed light on the varying possibilities and approaches of targeting Polβ for potential therapeutic intervention, we summarize the reported small molecule inhibitors of Polβ and discuss the genetic, biochemical and chemical studies that implicate additional options for Polβ inhibition. Further, we offer suggestions on possible inhibitor combinatorial approaches and the potential for tumor specificity for Polβ-inhibitors. PMID:22122465

  13. Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles.

    PubMed

    Lai, En Yin; Martinka, Peter; Fähling, Michael; Mrowka, Ralf; Steege, Andreas; Gericke, Adrian; Sendeski, Mauricio; Persson, P B; Persson, A Erik G; Patzak, Andreas

    2006-11-10

    Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II-induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II-induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II-induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney. PMID:17038642

  14. Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

    PubMed Central

    García-Sáinz, J A; Torner, M L

    1985-01-01

    Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected. PMID:3004405

  15. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2015-07-01

    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism.

  16. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2015-07-01

    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism. PMID:25910812

  17. Rapid Induction of Ion Pulses in Tomato, Cucumber, and Maize Plants following a Foliar Application of L(+)-Adenosine.

    PubMed Central

    Ries, S.; Savithiry, S.; Wert, V.; Widders, I.

    1993-01-01

    Application of picomole quantities of (+)-adenosine, a plant growth-regulating second messenger elicited by triacontanol, to tomato (Lycopersicon esculentum Mill.), maize (Zea mays L.), and cucumber (Cucumis sativa L.) foliage, increased Ca2+, Mg2+, and K+ concentrations in the exudate from the stumps of excised plants by 20 to 60% within 5 s after treatment. The change in ionic concentration of the exudate was transitory. When L(+)-adenosine and triacontanol were applied to different tomato plants at the same time, the L(+)-adenosine caused an increase in Ca2+ flux within 3 s, whereas a significant increase from triacontanol was not detectable until 5 min after application. This was expected because triacontanol elicits the formation of L(+)-adenosine. The enantiomer of L(+)-adenosine, D(-)-adenosine, had no effect on the cation concentration in tomato and inhibited the effect of L(+)-adenosine at equimolar or lower concentrations. These observations suggest that L(+)-adenosine acts by eliciting a rapidly propagated signal that increases the concentration of several ions in the apoplast. We postulate that modulations in apoplastic ion concentration, especially increases in Ca2+ concentration, constitute a mechanism by which plants regulate metabolic activity and growth in response to certain stimuli. PMID:12231664

  18. Binary Drugs: Conjugates of Purines and a Peptide That Bind to Both Adenosine and Substance P Receptors

    PubMed Central

    Jacobson, Kenneth A.; Lipkowski, Andrzej W.; Moody, Terry W.; Padgett, William; Pijl, Evelyn; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    A “functionalized congener” approach to adenosine receptor antagonists has provided a means to synthesize highly potent peptide conjugates of 1,3-dialkylxanthines. The antagonist XAC, such a functionalized xanthine amine congener, has been attached to a segment derived from the neurotransmitter peptide substance P (SP) to form a binary drug that binds to both receptors with Ki values of 35 nM (central A1-adenosine) and 300 nM (striatal SP). Coupling of the functionalized adenosine agonist N6-[p-(carboxymethyl)phenyl]adenosine to an SP C-terminal peptide also resulted in a binary drug that binds to both receptors. The demonstration that the biochemical properties of two unrelated drugs, both of which act through binding at extracellular receptors, may be combined in the same molecule suggests a novel strategy for drug design. In principle, a combined effect of the two different substances that produce the same final effect (e.g., hypotension by adenosine agonists and by SP analogues) might occur in vivo. Adenosine analogues have analgesic properties, and the binary drug derived from substance P and adenosine agonists or antagonists might provide useful tools for probing interrelationships of SP pathways and sites for the antinociceptive action of adenosine. PMID:2441057

  19. In vitro replication slippage by DNA polymerases from thermophilic organisms.

    PubMed

    Viguera, E; Canceill, D; Ehrlich, S D

    2001-09-14

    Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.

  20. Directed evolution of polymerase function by compartmentalized self-replication

    PubMed Central

    Ghadessy, Farid J.; Ong, Jennifer L.; Holliger, Philipp

    2001-01-01

    We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication. PMID:11274352

  1. Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

    PubMed

    Esyunina, D M; Kulbachinskiy, A V

    2015-10-01

    The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

  2. Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

    PubMed

    Cowart, M; Lee, C H; Gfesser, G A; Bayburt, E K; Bhagwat, S S; Stewart, A O; Yu, H; Kohlhaas, K L; McGaraughty, S; Wismer, C T; Mikusa, J; Zhu, C; Alexander, K M; Jarvis, M F; Kowaluk, E A

    2001-01-01

    The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

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

    PubMed Central

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

    2012-01-01

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

  4. Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70.

    PubMed

    Cheeseman, Matthew D; Westwood, Isaac M; Barbeau, Olivier; Rowlands, Martin; Dobson, Sarah; Jones, Alan M; Jeganathan, Fiona; Burke, Rosemary; Kadi, Nadia; Workman, Paul; Collins, Ian; van Montfort, Rob L M; Jones, Keith

    2016-05-26

    HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner. PMID:27119979

  5. Characterization of nitrobenzylthioinosine binding to nucleoside transport sites selective for adenosine in rat brain

    SciTech Connect

    Geiger, J.D.; LaBella, F.S.; Nagy, J.I.

    1985-03-01

    Nucleoside transport sites in rat brain membrane preparations were labeled with (/sup 3/H)nitrobenzylthioinosine ((/sup 3/H) NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with K/sub D/ and B/sub max/ values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of (/sup 3/H)NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The K/sub i/ values (micromolar concentration) of (/sup 3/H)NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parallel displacement curves indicating their interaction with a common site labeled by (/sup 3/H)NBI. The nucleobases, hypoxanthine and adenine, exhibited K/sub i/ values of 220 and 3640 microM, respectively. Adenosine receptor agonists exhibited moderate affinities for the (/sup 3/H)NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The K/sub i/ values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 microM, respectively. These affinities and the rank order of potencies indicate that (/sup 3/H)NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The K/sub i/ values of other nucleoside transport inhibitors were: nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 microM; and 2'-deoxyadenosine, 22 microM. These results indicate that (/sup 3/H)NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control adenosinergic function.

  6. L-nucleoside analogues as potential antimalarials that selectively target Plasmodium falciparum adenosine deaminase.

    PubMed

    Brown, D M; Netting, A G; Chun, B K; Choi, Y; Chu, C K; Gero, A M

    1999-01-01

    The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, Ki 7 pM; L-coformycin, Ki 250 pM). While the L-nucleoside analogues, L-adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino-1-(L-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.

  7. 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.

  8. 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

  9. Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3

    PubMed Central

    Nakel, Katharina; Bonneau, Fabien; Eckmann, Christian R.; Conti, Elena

    2015-01-01

    The Caenorhabditis elegans germ-line development defective (GLD)-2–GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2–GLD-3 acts by extending the short poly(A) tail of germ-line–specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3′ end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3′ end features of dormant cytoplasmic mRNAs. PMID:26124149

  10. Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

    PubMed Central

    Burak, Matthew J.; Guja, Kip E.; Garcia-Diaz, Miguel

    2015-01-01

    8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases. PMID:26220180

  11. Sperm motility and kinetics of dynein ATPase in astheno- and normozoospermic samples after stimulation with adenosine and its analogues.

    PubMed

    Romac, P; Zanić-Grubisić, T; Culić, O; Cvitković, P; Flogel, M

    1994-08-01

    We tested the effects of adenosine and 2-deoxyadenosine on the activation of human spermatozoa. In the asthenozoospermic group of patients adenosine produces an increase in sperm motility from 33.3 +/- 2.1% to 42.1 +/- 3.4%, progressive motility from 22.5 +/- 1.3% to 28.6 +/- 1.7% and forward progression rating from 2.1 +/- 0.2% to 2.8 +/- 0.1%. 2-Deoxyadenosine stimulated asthenozoospermic samples to a greater degree than adenosine. Sperm motility rose to 48.9 +/- 3.4%, progressive motility to 32.1 +/- 3.4% and forward progression rating to 3.0 +/- 0.1% following stimulation with 2-deoxy-adenosine. The kinetic parameters and basic characteristics of dynein ATPase were determined. The maximum activity of dynein ATPase, Vmax, was significantly different (P < 0.001) for asthenozoospermic and normozoospermic samples: 6.46 +/- 2.1 nmol Pi/mg/min and 16.99 +/- 3.7 nmol Pi/mg/min respectively. However, the enzyme affinity for ATP was not different. Stimulation of asthenozoospermic samples with adenosine and 2-deoxyadenosine caused an increase of Vmax (70-90% and 90-110% respectively) and no significant change in KM was observed. In order to block the nucleoside transporter and to eliminate the action of adenosine inside the cell, dipyridamole was used but the effects of adenosine were not neutralized. 5'-(N-ethylcarboxy-amido)-adenosine showed effects similar to those of adenosine, even when applied in 1 microM concentration. These results indicate that adenosine and its analogues stimulate sperm motility and activity of dynein ATPase, most probably via A2 receptors.

  12. Chaperoning of the A1-Adenosine Receptor by Endogenous Adenosine—An Extension of the Retaliatory Metabolite Concept*

    PubMed Central

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

    2015-01-01

    Cell-permeable orthosteric ligands can assist folding of G protein–coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y288 A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y288 A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress. PMID:25354767

  13. Activation of adenosine A1 receptors reduces anxiety-like behavior during acute ethanol withdrawal (hangover) in mice.

    PubMed

    Prediger, Rui D S; da Silva, George E; Batista, Luciano C; Bittencourt, Alvorita L; Takahashi, Reinaldo N

    2006-10-01

    Elevated signs of anxiety are observed in both humans and rodents during withdrawal from chronic as well as acute ethanol exposure, and it represents an important motivational factor for ethanol relapse. Several reports have suggested the involvement of brain adenosine receptors in different actions produced by ethanol such as motor incoordination and hypnotic effects. In addition, we have recently demonstrated that adenosine A1 receptors modulate the anxiolytic-like effect induced by ethanol in mice. In the present study, we evaluated the potential of adenosine A1 and A2A receptor agonists in reducing the anxiety-like behavior during acute ethanol withdrawal (hangover) in mice. Animals received a single intraperitoneal administration of saline or ethanol (4 g/kg) and were tested in the elevated plus maze after an interval of 0.5-24 h. The results indicated that hangover-induced anxiety was most pronounced between 12 and 18 h after ethanol administration, as indicated by a significant reduction in the exploration of the open arms of the maze. At this time interval, ethanol was completely cleared. The acute administration of 'nonanxiolytic' doses of adenosine and the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), but not the adenosine A2A receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA), at the onset of peak withdrawal (18 h), reduced this anxiogenic-like response. In addition, the effect of CCPA on the anxiety-like behavior of ethanol hangover was reversed by pretreatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results reinforce the notion of the involvement of adenosine receptors in the anxiety-like responses and indicate the potential of adenosine A1 receptor agonists to reduce the anxiogenic effects during ethanol withdrawal.

  14. Guanine-rich sequences inhibit proofreading DNA polymerases

    PubMed Central

    Zhu, Xiao-Jing; Sun, Shuhui; Xie, Binghua; Hu, Xuemei; Zhang, Zunyi; Qiu, Mengsheng; Dai, Zhong-Min

    2016-01-01

    DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment. PMID:27349576

  15. The Closing Mechanism of DNA Polymerase I at Atomic Resolution.

    PubMed

    Miller, Bill R; Beese, Lorena S; Parish, Carol A; Wu, Eugene Y

    2015-09-01

    DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl. PMID:26211612

  16. Inhibition of RNA polymerase by streptolydigin: no cycling allowed.

    PubMed

    Kyzer, Scotty; Zhang, Jinwei; Landick, Robert

    2005-08-26

    Bacterial RNA polymerase is a common target for many antibiotics. In two recent papers in Cell and Molecular Cell, and describe a structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin. Streptolydigin may prevent distortion of a "bridge" alpha helix postulated to occur during the nucleotide addition cycle of RNA polymerase or may block a small movement of the bridge helix that helps load nucleotide triphosphates into the active site. PMID:16122417

  17. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  18. Polymerase Chain Reaction on a Viral Nanoparticle.

    PubMed

    Carr-Smith, James; Pacheco-Gómez, Raúl; Little, Haydn A; Hicks, Matthew R; Sandhu, Sandeep; Steinke, Nadja; Smith, David J; Rodger, Alison; Goodchild, Sarah A; Lukaszewski, Roman A; Tucker, James H R; Dafforn, Timothy R

    2015-12-18

    The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years, much work has been carried out using a range of biomolecular chassis including α-helical coiled coils, β-sheet amyloids and even viral particles. In this work, we show how hybrid bionanoparticles can be produced from a viral M13 bacteriophage scaffold through conjugation with DNA primers that can template a polymerase chain reaction (PCR). This unprecedented example of a PCR on a virus particle has been studied by flow aligned linear dichroism spectroscopy, which gives information on the structure of the product as well as a new protototype methodology for DNA detection. We propose that this demonstration of PCR on the surface of a bionanoparticle is a useful addition to ways in which hybrid assemblies may be constructed using synthetic biology.

  19. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  20. RNA Polymerase Pausing during Initial Transcription.

    PubMed

    Duchi, Diego; Bauer, David L V; Fernandez, Laurent; Evans, Geraint; Robb, Nicole; Hwang, Ling Chin; Gryte, Kristofer; Tomescu, Alexandra; Zawadzki, Pawel; Morichaud, Zakia; Brodolin, Konstantin; Kapanidis, Achillefs N

    2016-09-15

    In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA. PMID:27618490

  1. RNA Polymerase Pausing during Initial Transcription.

    PubMed

    Duchi, Diego; Bauer, David L V; Fernandez, Laurent; Evans, Geraint; Robb, Nicole; Hwang, Ling Chin; Gryte, Kristofer; Tomescu, Alexandra; Zawadzki, Pawel; Morichaud, Zakia; Brodolin, Konstantin; Kapanidis, Achillefs N

    2016-09-15

    In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

  2. High-Throughput Polymerase Fidelity Evolution in Microfluidic Droplets

    NASA Astrophysics Data System (ADS)

    Collins, Jesse; de Paz, Alexandra; Cybulski, Ted; Bhan, Namita; Zhang, Huidan; Weitz, Dave; Tyo, Keith; Kording, Konrad

    Polymerases are technologically important as a tool in molecular biology, and are scientifically important for their role in DNA replication and inheritance. We study large numbers (at least millions) of polymerase mutants by compartmentalizing each gene in a droplet in a microfluidic device. Also in each droplet are in vitro transcription and translation proteins, such that mutant polymerases can be generated to extend their own gene along a known DNA template. Reading the resulting sequence tells us both the mutant gene sequence and the number of and particular errors that the resulting mutant polymerase made during extension. This work is supported by the NIH.

  3. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

    PubMed

    Bernad, A; Zaballos, A; Salas, M; Blanco, L

    1987-12-20

    The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.

  4. DNA replication. A familiar ring to DNA polymerase processivity.

    PubMed

    Wyman, C; Botchan, M

    1995-04-01

    Structural similarity reveals that prokaryotic and eukaryotic DNA polymerases share a mechanism for processivity--but the conservation of additional chromosomal replication mechanisms remains to be determined.

  5. Modification of RNA polymerase IIO subspecies after poliovirus infection.

    PubMed Central

    Rangel, L M; Fernandez-Tomas, C; Dahmus, M E; Gariglio, P

    1987-01-01

    Infection of HeLa cells with poliovirus results in a shutdown of host transcription. In an effort to understand the mechanism(s) that underlies this process, we analyzed the distribution of RNA polymerase IIO before and after viral infection. Analysis of free and chromatin-bound enzyme indicated that there is a significant reduction in RNA polymerase IIO following infection. This observation, together with increasing evidence that transcription is catalyzed by RNA polymerase IIO, supports the hypothesis that poliovirus-induced inhibition of host transcription occurs at the level of RNA chain initiation and involves the direct modification of RNA polymerase II. Images PMID:3029396

  6. Substrate-induced DNA polymerase β activation.

    PubMed

    Beard, William A; Shock, David D; Batra, Vinod K; Prasad, Rajendra; Wilson, Samuel H

    2014-11-01

    DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain. PMID:25261471

  7. A Cross-chiral RNA Polymerase Ribozyme

    PubMed Central

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer.1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes prior to the origin of RNA-based life.3,4 Once an RNA enzyme arose that could catalyze the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D-and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness. Such a cross-chiral RNA polymerase has now been developed using in vitro evolution. The D-RNA enzyme, consisting of 83 nucleotides, catalyzes the joining of L-mono- or oligonucleotide substrates on a complementary L-RNA template, and similarly for the L-enzyme with D-substrates and a D-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme's activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides. PMID:25363769

  8. Analgesic effects of adenosine in syndrome X are counteracted by theophylline: a double-blind placebo-controlled study.

    PubMed

    Eriksson, B E; Sadigh, B; Svedenhag, J; Sylvén, C

    2000-01-01

    It has been proposed that adenosine mediates ischaemic pain in humans. Patients with cardiac Syndrome X are hypersensitive to potential pain stimuli, including adenosine. On the other hand, recent findings suggest that low-dose adenosine infusion may have analgesic effects. Our aim was to test two hypotheses: (1) that the analgesic effect of adenosine is peripheral in origin, and (2) that part of the hypersensitivity to pain of patients with cardiac Syndrome X results from a disturbed mechanism of adenosine analgesia. A total of 12 female Syndrome X patients and eight healthy age-matched female controls were studied in a randomized, double-blind and placebo-controlled study. Adenosine (70 microg/min) or placebo was infused into the forearm via an intra-arterial catheter. After 15 min of infusion, a tourniquet on the upper arm was inflated to 225 mmHg to ensure arterial occlusion. The patient then carried out dynamic handgrip work at 60 Hz. Pain or discomfort in the forearm was estimated continuously according to the Borg CR-10 scale. After the first test, theophylline was infused for 10 min intravenously at a dose of 5 mg/kg body weight. The ischaemic forearm test was then repeated. On a second occasion, the procedure was repeated with the opposite treatment (adenosine/placebo). Only six of 12 Syndrome X patients completed the protocol because of pain during the catheterization procedure or an inability to establish an intra-arterial line. The time to onset of pain in the working, ischaemic forearm was greater for subjects treated with adenosine than for those treated with placebo, both in those Syndrome X patients who tolerated catheterization (49+/-27 s compared with 32+/-18 s; P<0.03) and in healthy controls (40+/-19 s compared with 16+/-8 s; P<0.02). The time to maximum pain, limiting ischaemic work, was also greater with adenosine pretreatment both in Syndrome X patients (137+/-28 s compared with 106+/-28 s; P<0.03) and in healthy controls (109+/-31 compared

  9. Brain stem adenosine receptors modulate centrally mediated hypotensive responses in conscious rats: A review.

    PubMed

    Nassar, Noha N; Abdel-Rahman, Abdel A

    2015-05-01

    Adenosine is implicated in the modulation of cardiovascular responses either at the peripheral or at central level in experimental animals. However, there are no dedicated reviews on the involvement of adenosine in mediating the hypotensive response of centrally administered clonidine in general and specifically in aortically barodenervated rats (ABD). The conscious ABD rat model exhibits surgically induced baroreflex dysfunction and exaggerated hypotensive response, compared with conscious sham-operated (SO) rats. The current review focuses on, the role of adenosine receptors in blood pressure (BP) regulation and their possible crosstalk with other receptors e.g. imidazoline (I1) and alpha (α2A) adrenergic receptor (AR). The former receptor is a molecular target for clonidine, whose hypotensive effect is enhanced approx. 3-fold in conscious ABD rats. We also discussed how the balance between the brain stem adenosine A1 and A2A receptors is regulated by baroreceptors and how such balance influences the centrally mediated hypotensive responses. The use of the ABD rat model yielded insight into the downstream signaling cascades following clonidine-evoked hypotension in a surgical model of baroreflex dysfunction. PMID:26257930

  10. Role of endogenous adenosine as a modulator of the renin response to salt restriction.

    PubMed

    Kuan, C J; Wells, J N; Jackson, E K

    1987-01-01

    Numerous studies indicate that exogenous adenosine can inhibit renin release. However, the hypothesis that endogenous adenosine functions to restrain the renin response to physiological and/or pharmacological stimuli remains untested. To address this hypothesis, we examined the effects of a novel adenosine receptor antagonist, 1,3-dipropyl-8-para-sulfophenylxanthine (DPSPX), on renin release in rats on a normal versus a low salt diet. DPSPX did not affect renal blood flow, glomerular filtration rate, filtration fraction, urine volume, or sodium excretion in rats on either a normal or low salt diet. In contrast, in rats on a low salt diet, DPSPX significantly increased arterial and renal venous plasma renin activity and the gradient of plasma renin activity across the kidney. DPSPX did not alter these indices of renin release in rats on a normal salt diet. These data support the hypothesis that endogenous adenosine functions to restrain the renin response to salt depletion. Finally, if these findings are applicable to man, caffeine consumption could account for the variable antihypertensive effect of a low salt diet. PMID:3330576

  11. Purine nucleoside metabolism in the erythrocytes of patients with adenosine deaminase deficiency and severe combined immunodeficiency.

    PubMed Central

    Agarwal, R P; Crabtree, G W; Parks, R E; Nelson, J A; Keightley, R; Parkman, R; Rosen, F S; Stern, R C; Polmar, S H

    1976-01-01

    Deficiency of erythrocytic and lymphocytic adenosine deaminase (ADA) occurs in some patients with severe combined immunodeficiency disease (SCID). SCID with ADA deficiency is inherited as an autosomal recessive trait. ADA is markedly reduced or undetectable in affected patients (homozygotes), and approximately one-half normal levels are found in individuals heterozygous for ADA deficiency. The metabolism of purine nucleosides was studied in erythrocytes from normal individuals, four ADA-deficiency patients, and two heterozygous individuals. ADA deficiency in intake erythrocytes was confirmed by a very sensitive ammonia-liberation technique. Erythrocytic ADA activity in three heterozygous individuals (0.07,0.08, and 0.14 mumolar units/ml of packed cells) was between that of the four normal controls (0.20-0.37 mumol/ml) and the ADA-deficient patients (no activity). In vitro, adenosine was incorporated principally into IMP in the heterozygous and normal individuals but into the adenosine nucleotides in the ADa-deficient patients. Coformycin (3-beta-D-ribofuranosyl-6,7,8-trihydroimidazo[4,5-4] [1,3] diazepin-8 (R)-ol), a potent inhibitor of ADA, made possible incorporation of adenosine nucleotides in the ADA-deficient patients... PMID:947948

  12. Gestational Diabetes Reduces Adenosine Transport in Human Placental Microvascular Endothelium, an Effect Reversed by Insulin

    PubMed Central

    Salomón, Carlos; Westermeier, Francisco; Puebla, Carlos; Arroyo, Pablo; Guzmán-Gutiérrez, Enrique; Pardo, Fabián; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

    2012-01-01

    Gestational diabetes mellitus (GDM) courses with increased fetal plasma adenosine concentration and reduced adenosine transport in placental macrovascular endothelium. Since insulin modulates human equilibrative nucleoside transporters (hENTs) expression/activity, we hypothesize that GDM will alter hENT2-mediated transport in human placental microvascular endothelium (hPMEC), and that insulin will restore GDM to a normal phenotype involving insulin receptors A (IR-A) and B (IR-B). GDM effect on hENTs expression and transport activity, and IR-A/IR-B expression and associated cell signalling cascades (p42/44 mitogen-activated protein kinases (p42/44mapk) and Akt) role in hPMEC primary cultures was assayed. GDM associates with elevated umbilical whole and vein, but not arteries blood adenosine, and reduced hENTs adenosine transport and expression. IR-A/IR-B mRNA expression and p42/44mapk/Akt ratios (‘metabolic phenotype’) were lower in GDM. Insulin reversed GDM-reduced hENT2 expression/activity, IR-A/IR-B mRNA expression and p42/44mapk/Akt ratios to normal pregnancies (‘mitogenic phenotype’). It is suggested that insulin effects required IR-A and IR-B expression leading to differential modulation of signalling pathways restoring GDM-metabolic to a normal-mitogenic like phenotype. Insulin could be acting as protecting factor for placental microvascular endothelial dysfunction in GDM. PMID:22808198

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

    PubMed Central

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

    2010-01-01

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

  14. Adenosine A2A receptor antagonists: blockade of adenosinergic effects and T regulatory cells

    PubMed Central

    Sitkovsky, M; Lukashev, D; Deaglio, S; Dwyer, K; Robson, S C; Ohta, A

    2008-01-01

    The intensity and duration of host responses are determined by protective mechanisms that control tissue injury by dampening down inflammation. Adenosine generation and consequent effects, mediated via A2A adenosine receptors (A2AR) on effector cells, play a critical role in the pathophysiological modulation of these responses in vivo. Adenosine is both released by hypoxic cells/tissues and is also generated from extracellular nucleotides by ecto-enzymes e.g. CD39 (ENTPD1) and CD73 that are expressed by the vasculature and immune cells, in particular by T regulatory cell. In general, these adenosinergic mechanisms minimize the extent of collateral damage to host tissues during the course of inflammatory reactions. However, induction of suppressive pathways might also cause escape of pathogens and permit dissemination. In addition, adenosinergic responses may inhibit immune responses while enhancing vascular angiogenic responses to malignant cells that promote tumor growth. Novel drugs that block A2AR-adenosinergic effects and/or adenosine generation have the potential to boost pathogen destruction and to selectively destroy malignant tissues. In the latter instance, future treatment modalities might include novel ‘anti-adenosinergic' approaches that augment immune clearance of malignant cells and block permissive angiogenesis. This review addresses several possible pharmacological modalities to block adenosinergic pathways and speculates on their future application together with impacts on human disease. PMID:18311159

  15. Phenylephrine stimulated breakdown of phosphoinositides in brown adipocytes is attenuated by adenosine

    SciTech Connect

    Schimmel, R.J.

    1986-03-01

    Selective activation of alpha adrenergic receptors on brown adipocytes brings about increased mitochondrial respiration. This response is associated with a rapid breakdown of phosphoinositides in the plasma membrane. The authors have shown that respiration increased by alpha receptor activation can be inhibited by adenosine but the mechanisms underlying this effect are unknown. The present study probes the possibility that adenosine inhibition of alpha receptor stimulated respiration is secondary to an inhibition of stimulated breakdown of inositol phospholipids. Phospholipids were labeled with (/sup 32/P) by incubation with (/sup 32/P)-Pi for up to four hours. Phenylephrine and other ligands were then added and the radioactivity present in individual lipids determined following their resolution by thin layer chromatography. Addition of 2-chloroadenosine or phenylisopropyl adenosine, but not 2',5'-dideoxyadenosine, inhibited phenylephrine promoted breakdown of phosphoinositides. The dose response relation for this effect was similar to that for attenuation of stimulated respiration. This finding demonstrates adenosine inhibition of a phospholipase in brown fat cells and suggests the possibility that breakdown of inositol phospholipids is a critical control site for stimulation and attenuation of respiration.

  16. Unexpected Discovery of Dichloroacetate Derived Adenosine Triphosphate Competitors Targeting Pyruvate Dehydrogenase Kinase To Inhibit Cancer Proliferation.

    PubMed

    Zhang, Shao-Lin; Hu, Xiaohui; Zhang, Wen; Tam, Kin Yip

    2016-04-14

    Pyruvate dehydrogenase kinases (PDKs) have recently emerged as an attractive target for cancer therapy. Herein, we prepared a series of compounds derived from dichloroacetate (DCA) which inhibited cancer cells proliferation. For the first time, we have successfully developed DCA derived inhibitors that preferentially bind to the adenosine triphosphate (ATP) pocket of PDK isoform 1 (PDK1).

  17. Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states.

    PubMed

    Little, Joshua W; Ford, Amanda; Symons-Liguori, Ashley M; Chen, Zhoumou; Janes, Kali; Doyle, Timothy; Xie, Jennifer; Luongo, Livio; Tosh, Dillip K; Maione, Sabatino; Bannister, Kirsty; Dickenson, Anthony H; Vanderah, Todd W; Porreca, Frank; Jacobson, Kenneth A; Salvemini, Daniela

    2015-01-01

    Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain. PMID:25414036

  18. Dynamic Regulation of the Adenosine Kinase Gene during Early Postnatal Brain Development and Maturation

    PubMed Central

    Kiese, Katharina; Jablonski, Janos; Boison, Detlev; Kobow, Katja

    2016-01-01

    The ubiquitous metabolic intermediary and nucleoside adenosine is a “master regulator” in all living systems. Under baseline conditions adenosine kinase (ADK) is the primary enzyme for the metabolic clearance of adenosine. By regulating the availability of adenosine, ADK is a critical upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. ADK protein exists in the two isoforms nuclear ADK-L, and cytoplasmic ADK-S, which are subject to dynamic expression changes during brain development and in response to brain injury; however, gene expression changes of the Adk gene as well as regulatory mechanisms that direct the cell-type and isoform specific expression of ADK have never been investigated. Here we analyzed potential gene regulatory mechanisms that may influence Adk expression including DNA promoter methylation, histone modifications and transcription factor binding. Our data suggest binding of transcription factor SP1 to the Adk promoter influences the regulation of Adk expression. PMID:27812320

  19. Adenosine Receptor Blockade by Caffeine Inhibits Carotid Sinus Nerve Chemosensory Activity in Chronic Intermittent Hypoxic Animals.

    PubMed

    Sacramento, J F; Gonzalez, C; Gonzalez-Martin, M C; Conde, S V

    2015-01-01

    Adenosine is a key excitatory neurotransmitter at the synapse between O(2)-sensing chemoreceptor cells-carotid sinus nerve (CSN) endings in the carotid body (CB). Herein, we have investigated the significance of adenosine, through the blockade of its receptors with caffeine, on the CB hypoxic sensitization induced by chronic intermittent hypoxia (CIH) in the rat. CIH animals were obtained by submitting rats during 15 days from 8:00 to 16:00 to 10 %O(2) for 40 s and 20 % O(2) for 80 s (i.e., 30 episodes/h). Caffeine (1 mM) was tested in spontaneous and 5 %O(2) evoked-CSN chemosensory activity in normoxic and CIH animals. CIH decreased basal spontaneous activity but increased significantly CSN activity evoked by acute hypoxia. Caffeine did not modify basal spontaneous activity in normoxic rats, but decreased significantly by 47.83 % basal activity in CIH animals. In addition, acute application of caffeine decreased 49.31 % and 56.01 % the acute hypoxic response in normoxic and CIH animals, respectively. We demonstrate that adenosine contributes to fix CSN basal activity during CIH, being also involved in hypoxic CB chemotransduction. It is concluded that adenosine participates in CB sensitization during CIH. PMID:26303475

  20. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships

    PubMed Central

    Cheong, Siew Lee; Venkatesan, Gopalakrishnan; Paira, Priyankar; Jothibasu, Ramasamy; Mandel, Alexander Laurence; Federico, Stephanie; Spalluto, Giampiero; Pastorin, Giorgia

    2011-01-01

    In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3) has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR) profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists. PMID:25954519

  1. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia

    PubMed Central

    Pedata, Felicita; Pugliese, Anna Maria; Coppi, Elisabetta; Dettori, Ilaria; Maraula, Giovanna; Cellai, Lucrezia; Melani, Alessia

    2014-01-01

    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke. PMID:25165414

  2. John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral activity.

    PubMed

    De Clercq, Erik

    2005-01-01

    Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626-629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c3 Ado), neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c3 Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c3Ado and 3-deazaneplanocin A should in thefirst place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola. PMID:16438025

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

    SciTech Connect

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

    1982-08-01

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

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

    SciTech Connect

    Jones, Anthony T.; Hartwig, Eric O.

    1982-08-01

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

  5. Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

    PubMed

    Klein, C

    1975-09-25

    Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity. PMID:170256

  6. NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization.

    PubMed

    Chow, W Ying; Rajan, Rakesh; Muller, Karin H; Reid, David G; Skepper, Jeremy N; Wong, Wai Ching; Brooks, Roger A; Green, Maggie; Bihan, Dominique; Farndale, Richard W; Slatter, David A; Shanahan, Catherine M; Duer, Melinda J

    2014-05-16

    Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach. PMID:24833391

  7. Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

    PubMed

    Brito, Rafael; Pereira-Figueiredo, Danniel; Socodato, Renato; Paes-de-Carvalho, Roberto; Calaza, Karin C

    2016-08-01

    Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30 mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48 h. Accordingly, caffeine (30 mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6 h after injection, but an increase after 18 and 24 h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24 h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48 h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an in vivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It

  8. Mechanisms involved in increased sensitivity to adenosine A(2A) receptor activation and hypoxia-induced vasodilatation in porcine coronary arteries.

    PubMed

    Hedegaard, Elise R; Nielsen, Berit D; Mogensen, Susie; Rembold, Christopher M; Frøbert, Ole; Simonsen, Ulf

    2014-01-15

    Hypoxia-induced coronary vasorelaxation is a compensatory mechanism increasing blood flow. We hypothesized that hypoxia shares pathways with adenosine and causes vasorelaxation through the adenosine A(2A) receptor and force suppression by increasing cAMP and phosphorylated heat shock protein (HSP)20. Adenosine receptors in porcine left anterior descending coronary arteries (LAD) were examined by RT-PCR and isometric tension recording in myographs. Vasorelaxation was induced by adenosine, 1% oxygen, or both in the absence or presence of ZM241385, an adenosine A(2A) receptor antagonist. cAMP was determined by ELISA and p-HSP20/HSP20 and p-MLC/MLC were determined by immunoblotting and densitometric analyses. In coronary arteries exposed to 1% oxygen, there was increased sensitivity to adenosine, the adenosine A2 selective agonist NECA, and the adenosine A(2A) selective receptor agonist CGS21680. ZM241385 shifted concentration-response curves for CGS21680 to the right, whereas the adenosine A1 antagonist DPCPX, the adenosine A2B receptor antagonist MRS1754 and the adenosine A3 receptor antagonist MRS1523 failed to reduce vasodilatation induced by CGS21680. 1% oxygen or adenosine increased cAMP accumulation and HSP20 phosphorylation without changing T850-MYPT1 and MLC phosphorylation. ZM241385 failed to change 1% oxygen-induced vasodilation, cAMP accumulation, HSP20 phosphorylation and MLC phosphorylation. The PKA inhibitor Rp-8-CPT-cAMPS significantly reduced vasorelaxation induced by 1% oxygen or CGS21680. Our findings suggest that the increased sensitivity to adenosine, NECA, and CGS21680 at 1% oxygen involves adenosine A(2A) receptors. Adenosine and 1% oxygen induce vasorelaxation in PGF2α-contracted porcine coronary arteries partly by force suppression caused by increased cAMP and phosphorylation of HSP20.

  9. RNA Polymerases of Maize. Purification and Molecular Structure of DNA-dependent RNA Polymerase II*

    PubMed Central

    Mullinix, Kathleen P.; Strain, Gustave C.; Bogorad, Lawrence

    1973-01-01

    Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE- and DNA-celluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase IIa is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000, 20,000, and 17,000. Images PMID:4525172

  10. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    SciTech Connect

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang; Yi, Kyu Yang; Chung, Hun-Jong; Park, Jong Seok; Kim, Bokyung; Feng, Zhong-Ping; Shin, Hwa-Sup

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  11. Effect of temozolomide on the viability of musculoskeletal sarcoma cells

    PubMed Central

    KUSABE, YUTA; KAWASHIMA, HIROYUKI; OGOSE, AKIRA; SASAKI, TARO; ARIIZUMI, TAKASHI; HOTTA, TETSUO; ENDO, NAOTO

    2015-01-01

    Musculoskeletal sarcomas (MSS) are a heterogeneous group of malignancies with relatively high mortality rates. The prognosis for patients with MSS is poor, with few drugs inducing measurable activity. Alkylating agents, namely ifosfamide and dacarbazine, which act nonspecifically on proliferating cells, are the typical therapy prescribed for advanced MSS. A novel alkylating agent, temozolomide (TMZ), has several advantages over existing alkylating agents. TMZ induces the formation of O6-methylguanine in DNA, thereby inducing mismatches during DNA replication and the subsequent activation of apoptotic pathways. However, due to conflicting data in the literature, the mechanism of TMZ action has remained elusive. Therefore, the present study aimed to evaluate apoptosis in MSS cells treated with TMZ, and to evaluate the correlation between TMZ action and survival pathways, including the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen activated protein kinase (MAPK) pathways. Cell proliferation was evaluated by performing an XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay. Apoptotic morphological changes, for example chromatin condensation, were evaluated by fluorescence confocal microscopy. The expression of the apoptosis-associated proteins caspase-3, poly adenosine diphosphate ribose polymerase (PARP), Akt and ERK1/2, was determined by western blotting. The results of the present study indicated that, in certain MSS cells, the IC50 value was lower than that in TMZ-sensitive U-87 MG cells. Furthermore, TMZ treatment was associated with apoptotic morphological changes and the expression levels of pro-apoptotic cleaved caspase-3 and PARP were also increased in TMZ-treated MSS cells. In addition, the results indicated that PI3K/Akt and ERK1/2 MAPK were constitutively phosphorylated in MSS cells, and phosphorylation of PI3K/Akt was suppressed in certain

  12. Efficacy of coronary fractional flow reserve using contrast medium compared to adenosine

    PubMed Central

    Tanboğa, Ibrahim Halil; Aksakal, Enbiya; Aksu, Uğur; Gulcu, Oktay; Birdal, Oğuzhan; Arısoy, Arif; Kalaycı, Arzu; Ulusoy, Fatih Rifat; Sevimli, Serdar

    2016-01-01

    Introduction Coronary fractional flow reserve (FFR) is recommended as the gold standard method in evaluating intermediate coronary stenoses. However, there are significant debates concerning the agents and the timing of the measurement. Aim To compare the contrast medium induced Pd/Pa ratio (CMR) with the FFR. Material and methods We enrolled 28 consecutive patients with 34 intermediate lesions who underwent coronary FFR measurement by intracoronary (i.c.) adenosine. After baseline Pd/Pa was calculated, a single contrast medium (Iomeron) injection of 6 ml (3 ml/s) was performed manually. Within 10 s after the contrast medium injection, the CMR was calculated. Bolus injection of i.c. adenosine was performed to induce maximal hyperemia (from 60 µg to 600 µg), and when it was ≤ 0.80, the intermediate lesion was considered as significant. Results After bolus i.c. adenosine, 12 lesions of 34 (35.3%) were identified as significant. The CMR value was 0.86 ±0.06 (range: 0.71–0.97). There were no significant differences between FFR and CMR values (p = 0.108). A substantial positive correlation between adenosine and contrast values was detected (0.886 and p < 0.001). Good agreement in Bland-Altman analysis was revealed (mean bias was 0.027, 95% confidence interval 0.038–0.092). Receiver operating characteristics curve analysis showed 90.9% sensitivity and 91.7% specificity for a cut-off value of 0.85 for the CMR compared to FFR (≤ 0.80). Conclusions Our study showed that measuring the CMR is a feasible method compared to FFR. The CMR may be used in situations where adenosine cannot be administered. PMID:27625683

  13. Adenosine, caffeine, and performance: from cognitive neuroscience of sleep to sleep pharmacogenetics.

    PubMed

    Urry, Emily; Landolt, Hans-Peter

    2015-01-01

    An intricate interplay between circadian and sleep-wake homeostatic processes regulate cognitive performance on specific tasks, and individual differences in circadian preference and sleep pressure may contribute to individual differences in distinct neurocognitive functions. Attentional performance appears to be particularly sensitive to time of day modulations and the effects of sleep deprivation. Consistent with the notion that the neuromodulator, adenosine , plays an important role in regulating sleep pressure, pharmacologic and genetic data in animals and humans demonstrate that differences in adenosinergic tone affect sleepiness, arousal and vigilant attention in rested and sleep-deprived states. Caffeine--the most often consumed stimulant in the world--blocks adenosine receptors and normally attenuates the consequences of sleep deprivation on arousal, vigilance, and attention. Nevertheless, caffeine cannot substitute for sleep, and is virtually ineffective in mitigating the impact of severe sleep loss on higher-order cognitive functions. Thus, the available evidence suggests that adenosinergic mechanisms, in particular adenosine A2A receptor-mediated signal transduction, contribute to waking-induced impairments of attentional processes, whereas additional mechanisms must be involved in higher-order cognitive consequences of sleep deprivation. Future investigations should further clarify the exact types of cognitive processes affected by inappropriate sleep. This research will aid in the quest to better understand the role of different brain systems (e.g., adenosine and adenosine receptors) in regulating sleep, and sleep-related subjective state, and cognitive processes. Furthermore, it will provide more detail on the underlying mechanisms of the detrimental effects of extended wakefulness, as well as lead to the development of effective, evidence-based countermeasures against the health consequences of circadian misalignment and chronic sleep restriction.

  14. The adenosine receptor affinities and monoamine oxidase B inhibitory properties of sulfanylphthalimide analogues.

    PubMed

    Van der Walt, Mietha M; Terre'Blanche, Gisella; Petzer, Anél; Petzer, Jacobus P

    2015-04-01

    Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties of a series of 4- and 5-sulfanylphthalimide analogues. Since adenosine antagonists (A1 and A2A subtypes) and MAO-B inhibitors are considered agents for the therapy of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs that antagonize adenosine receptors and inhibit MAO-B may have enhanced therapeutic value. The results document that the sulfanylphthalimide analogues are selective for the adenosine A1 receptor over the A2A receptor subtype, with a number of compounds also possessing MAO-B inhibitory properties. Among the compounds evaluated, 5-[(4-methoxybenzyl)sulfanyl]phthalimide was found to possess the highest binding affinity to adenosine A1 receptors with a Ki value of 0.369 μM. This compound is reported to also inhibit MAO-B with an IC50 value of 0.020 μM. Such dual-target-directed compounds may act synergistic in the treatment of Parkinson's disease: antagonism of the A1 receptor may facilitate dopamine release, while MAO-B inhibition may reduce dopamine metabolism. Additionally, dual-target-directed compounds may find therapeutic value in Alzheimer's disease: antagonism of the A1 receptor may be beneficial in the treatment of cognitive dysfunction, while MAO-B inhibition may exhibit neuroprotective properties. In neurological diseases, such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs are expected to be advantageous over single-target treatments.

  15. Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2014-02-01

    Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8μg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension. PMID:24216055

  16. Role of nitric oxide in adenosine-induced vasodilation in humans

    NASA Technical Reports Server (NTRS)

    Costa, F.; Biaggioni, I.; Robertson, D. (Principal Investigator)

    1998-01-01

    Vasodilation is one of the most prominent effects of adenosine and one of the first to be recognized, but its mechanism of action is not completely understood. In particular, there is conflicting information about the potential contribution of endothelial factors. The purpose of this study was to explore the role of nitric oxide in the vasodilatory effect of adenosine. Forearm blood flow responses to intrabrachial adenosine infusion (125 microg/min) were assessed with venous occlusion plethysmography during intrabrachial infusion of saline or the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) (12.5 mg/min). Intrabrachial infusions of acetylcholine (50 microg/min) and nitroprusside (3 microg/min) were used as a positive and negative control, respectively. These doses were chosen to produce comparable levels of vasodilation. In a separate study, a second saline infusion was administered instead of L-NMMA to rule out time-related effects. As expected, pretreatment with L-NMMA reduced acetylcholine-induced vasodilation; 50 microg/min acetylcholine increased forearm blood flow by 150+/-43% and 51+/-12% during saline and L-NMMA infusion, respectively (P<.01, n=6). In contrast, L-NMMA did not affect the increase in forearm blood flow produced by 3 microg/min nitroprusside (165+/-30% and 248+/-41% during saline and L-NMMA, respectively) or adenosine (173+/-48% and 270+/-75% during saline and L-NMMA, respectively). On the basis of our observations, we conclude that adenosine-induced vasodilation is not mediated by nitric oxide in the human forearm.

  17. Modulation of audiogenic seizures by histamine and adenosine receptors in the inferior colliculus.

    PubMed

    Feng, H J; Faingold, C L

    2000-05-01

    Susceptibility to behaviorally similar audiogenic seizures (AGS) occurs genetically and is inducible during ethanol withdrawal (ETX). Comparisons between AGS mechanisms of genetically epilepsy-prone rats (GEPR-9s) and ethanol-withdrawn rats (ETX-Rs) are yielding information about general pathophysiological mechanisms of epileptogenesis. The inferior colliculus (IC) is the AGS initiation site. Excitatory amino acid (EAA) abnormalities in the IC are implicated in AGS, and histamine and adenosine receptor activation each reduce EAA release and inhibit several seizure types. Previous studies indicate that focal infusion of an adenosine receptor agonist into the IC blocked AGS in GEPR-9s, but the effects of adenosine receptor activation in the IC on AGS in ETX-Rs are unknown. The effects of histamine receptor activation on either form of AGS are also unexamined. The present study evaluated effects of histamine or a nonselective adenosine A(1) agonist, 2-chloroadenosine, on AGS by focal microinjection into the IC. Ethanol dependence and AGS susceptibility were induced in normal rats by intragastric ethanol. Histamine (40 or 60 nmol/side) significantly reduced AGS in GEPR-9s, but histamine in doses up to 120 nmol/side did not affect AGS in ETX-Rs. 2-Chloroadenosine (5 or 10 nmol/side) did not affect AGS in ETX-Rs, despite the effectiveness of lower doses of this agent in GEPR-9s reported previously. Thus, histamine and adenosine receptors in the IC modulate AGS of GEPR-9s, but do not modulate ETX-induced AGS. The reasons for this difference may involve the chronicity of AGS susceptibility in GEPR-9s, which may lead to more extensive neuromodulation as compensatory mechanisms to limit the seizures compared to the acute AGS of ETX-Rs.

  18. Adenosine decreases post-ischaemic cardiac TNF-alpha production: anti-inflammatory implications for preconditioning and transplantation.

    PubMed Central

    Meldrum, D R; Cain, B S; Cleveland, J C; Meng, X; Ayala, A; Banerjee, A; Harken, A H

    1997-01-01

    Tumour necrosis factor-alpha (TNF-alpha) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischaemia-reperfusion injury (I/R), sepsis, chronic heart failure and cardiac allograft rejection. Cardiac resident macrophages, infiltrating leucocytes, and cardiomyocytes themselves produce TNF-alpha. Although adenosine reduces macrophage TNF-alpha production and protects myocardium against I/R, it remains unknown whether I/R induces an increase in cardiac TNF-alpha in a crystalloid-perfused model (in the absence of blood), and, whether adenosine decreases cardiac TNF-alpha and protects function after I/R. To study this, isolated rat hearts were crystalloid-perfused using the Langendorff method and subjected to I/R, with or without adenosine pretreatment. Post-ischaemic cardiac TNF-alpha (enzyme-linked immunosorbent assay and bioassay) and function were determined (Langendorff). I/R increased cardiac TNF-alpha and impaired myocardial function. Adenosine decreased cardiac TNF-alpha and improved post-ischaemic functional recovery. This study demonstrates that: first, I/R induces an increase in cardiac tissue TNF-alpha in a crystalloid-perfused model: second, adenosine decreases cardiac TNF-alpha and improves post-ischaemic myocardial function; third, decreased cardiac TNF-alpha may represent a mechanism by which adenosine protects myocardium; and fourth, adenosine-induced suppression of cardiac TNF-alpha may provide an anti-inflammatory link to preconditioning and have implications for cardiac allograft preservation. PMID:9497488

  19. Electrochemical aptasensor for the detection of adenosine by using PdCu@MWCNTs-supported bienzymes as labels.

    PubMed

    Wu, Dan; Ren, Xiang; Hu, Lihua; Fan, Dawei; Zheng, Yang; Wei, Qin

    2015-12-15

    A highly sensitive electrochemical adenosine aptasensor was fabricated by covalently immobilizing 3'-NH2-terminated capture probe (SSDNA1) and thionine (TH) on Au-GS modified glassy carbon electrode. 3'-SH-terminated adenosine aptamer (SSDNA2) was adsorbed onto palladium/copper alloyed supported on MWCNTs (PdCu@MWCNTs)-conjugated multiple bienzymes, glucose oxidase (GOx), and horseradish peroxidase (HRP) (SSDNA2/PdCu@MWCNTs/HRP/GOx). Then, it was immobilized onto the electrode surface through the hybridization between the adenosine aptamer and the capture probe. The signal was amplified based on the gradual electrocatalytic reduction of GOx-generated hydrogen peroxide by the multiple HRP through the mediating ability of the loaded multiple TH. However, the peak current of TH decreased in the presence of adenosine because the interaction between adenosine and its aptamer made SSDNA2/PdCu@MWCNTs/HRP/GOx release from the modified electrode. Various experimental parameters have been optimized for the detection of adenosine and tests for selectivity, reproducibility and stability have also been performed. Under the optimal condition, the proposed aptasensor displayed a wide linear range (10-400 nM) with the low detection limit (2.5 nM), which has been applied in human serum samples with satisfactory results. Thus, the combination of Au-GS as a sensor platform and PdCu@MWCNTs/HRP/GOx as labels can be a promising amplification strategy for highly sensitive adenosine detection. PMID:26164010

  20. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

    PubMed Central

    IJzerman, Adriaan P.; Jacobson, Kenneth A.; Linden, Joel; Müller, Christa E.

    2011-01-01

    In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited. PMID:21303899

  1. Differences in adenosine A-1 and A-2 receptor density revealed by autoradiography in methylxanthine-sensitive and insensitive mice

    SciTech Connect

    Jarvis, M.F.; Williams, M.

    1988-07-01

    Two strains of inbred mice, CBA/J and SWR/J, have been identified which are, respectively, sensitive and insensitive to the behavioral and toxic effects of methylxanthines. Autoradiographic analyses of brain adenosine receptors were conducted with (/sup 3/H)CHA to label adenosine A-1 receptors and (/sup 3/H)NECA, in the presence of 50 nM CPA, to label adenosine A-2 receptors. For both mouse strains, adenosine A-1 receptors were most highly concentrated in the hippocampus and cerebellum whereas adenosine A-2 receptors were selectively localized in the striatum. CBA/J mice displayed a 30% greater density of adenosine A-1 receptors in the hippocampal CA-1 and CA-3 regions and in the cerebellum as compared to the SWR/J mice. The number of A-2 receptors (Bmax) was 40% greater in the striatum and olfactory tubercle of CBA/J as compared to SWR/J mice. No significant regional differences in A-1 or A-2 receptor affinities were observed between these inbred strains of mice. These results indicate that the differential sensitivity to methylxanthines between these mouse strains may reflect a genetically mediated difference in regional adenosine receptor densities.

  2. Activation of nicotinic ACh receptors with α4 subunits induces adenosine release at the rat carotid body

    PubMed Central

    Conde, Sílvia V; Monteiro, Emília C

    2006-01-01

    The effect of ACh on the release of adenosine was studied in rat whole carotid bodies, and the nicotinic ACh receptors involved in the stimulation of this release were characterized. ACh and nicotinic ACh receptor agonists, cytisine, DMPP and nicotine, caused a concentration-dependent increase in adenosine production during normoxia, with nicotine being more potent and efficient in stimulating adenosine release from rat CB than cytisine and DMPP. D-Tubocurarine, mecamylamine, DHβE and α-bungarotoxin, nicotinic ACh receptor antagonists, caused a concentration-dependent reduction in the release of adenosine evoked by hypoxia. The rank order of potency for nicotinic ACh receptor antagonists that inhibit adenosine release was DHβE>mecamylamine>D-tubocurarine>α-bungarotoxin. The effect of the endogenous agonist, ACh, which was mimicked by nicotine, was antagonized by DHβE, a selective nicotinic receptor antagonist. The ecto-5′-nucleotidase inhibitor AOPCP produces a 72% inhibition in the release of adenosine from CB evoked by nicotine. Taken together, these data indicate that ACh induced the production of adenosine, mainly from extracellular ATP catabolism at the CB through a mechanism that involves the activation of nicotinic receptors with α4 and β2 receptor subunits. PMID:16444287

  3. Binding induced colocalization activated hybridization chain reaction on the surface of magnetic nanobead for sensitive detection of adenosine.

    PubMed

    Feng, Chunjing; Hou, Zhun; Jiang, Wei; Sang, Lihong; Wang, Lei

    2016-12-15

    Herein, a sensitive and enzyme-free assay for adenosine detection has been developed on the basis of binding induced colocalization activated hybridization chain reaction (HCR) strategy on the surface of magnetic nanobead. First, the recognition probe was fabricated and divided into two parts: the Apt-1 that composed a part of adenosine aptamer and toehold domain, and the Apt-2 that consisted of another part of adenosine aptamer and branch migration domain. The Apt-1 was immobilized on a streptavidin-magnetic nanobead (streptavidin-MNBs) that played the roles of enrichment and separation. Then the recognition event of adenosine could bring the two parts of aptamer together and induce the colocalization of toehold domain and branch migration domain, which could serve as an integrated initiator to trigger the HCR, producing a long nicked double-stranded polymer. Finally, the intercalating dye SYBR Green I was inserted into the polymer, generating an enhanced fluorescence signal. In this strategy, the initiator was divided into two parts and could be suppressed effectively in the absence of adenosine. Utilizing the separated function, the spontaneous hybridization of H1 and H2 could be avoided, and a low background could be acquired. Moreover, through the double amplification of HCR and multimolecules binding of SYBR Green I, highly sensitive and enzyme-free detection were achieved. The detection limit for adenosine detection was 2.0×10(-7)mol/L, which was comparable or superior to the previous aptasensors. Importantly, adenosine analysis in human urines has been performed, and this strategy could significantly distinguish the adenosine content in normal human urines and cancer patient urines, suggesting that this proposed assay will become a reliable and sensitive adenosine detection method in early clinical diagnosis and medical research.

  4. GLUCOSE-INDUCED INTESTINAL VASODILATION VIA ADENOSINE A1 RECEPTORS REQUIRES NITRIC OXIDE BUT NOT K+ATP CHANNELS

    PubMed Central

    Matheson, Paul J.; Li, Na; Harris, Patrick D.; Zakaria, El Rasheid; Garrison, R. Neal

    2010-01-01

    INTRODUCTION Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that ATP utilization during glucose absorption would increase adenosine metabolite release, which acts on adenosine A1 receptors to alter endothelial production of NO and/or activate ATP-dependent potassium channels (K+ATP) to dilate intestinal microvessels. METHODS Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles and proximal (p3A) and distal (d3A) premucosal arterioles during exposure to isotonic glucose or mannitol solutions alone or in the presence of the selective nitric oxide synthase (NOS) inhibitor (L-NMMA), an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine (DPCPX)), or a K+ATP channel inhibitor (glibenclamide). RESULTS As expected, glucose exposure caused rapid dilation of both p3A and d3A arterioles, while mannitol exposure had no effect on microvascular diameters. Adenosine A1 receptor blockade completely prevented glucose-induced dilation of the premucosal arterioles. NOS inhibition significantly blunted the glucose-induced vasodilation of the premucosal arterioles, but had little effect in the mannitol group. Simultaneous application of both the NOS inhibitor and the adenosine A1 receptor antagonist gave the same reduction in glucose-induced dilation of the premucosal arterioles as the adenosine A1 receptor antagonist alone. Blockade of K+ATP channels with glibenclamide did not attenuate glucose-induced vasodilation of the premucosal arterioles. CONCLUSIONS These data suggest that glucose-induced vasodilation of premucosal jejunal arterioles is mediated through adenosine A1 receptors, and NO at least partially mediates the adenosine A1 receptor-induced vasodilation. In addition, K+ATP channels are not involved in premucosal arteriolar vasodilation during intestinal glucose exposure. PMID

  5. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    SciTech Connect

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

  6. Platelet-derived growth factor receptor-α-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles.

    PubMed

    Kurahashi, Masaaki; Mutafova-Yambolieva, Violeta; Koh, Sang Don; Sanders, Kenton M

    2014-09-15

    Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, β-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRα(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.

  7. Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

    PubMed

    Podestá, Dolores; García-Herreros, María I; Cannata, Joaquín J B; Stoppani, Andrés O M; Fernández Villamil, Silvia H

    2004-06-01

    Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

  8. Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

    PubMed

    Sharma, Amit; Leach, Robert N; Gell, Christopher; Zhang, Nan; Burrows, Patricia C; Shepherd, Dale A; Wigneshweraraj, Sivaramesh; Smith, David Alastair; Zhang, Xiaodong; Buck, Martin; Stockley, Peter G; Tuma, Roman

    2014-04-01

    Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase. PMID:24553251

  9. Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies

    PubMed Central

    Sharma, Amit; Leach, Robert N.; Gell, Christopher; Zhang, Nan; Burrows, Patricia C.; Shepherd, Dale A.; Wigneshweraraj, Sivaramesh; Smith, David Alastair; Zhang, Xiaodong; Buck, Martin; Stockley, Peter G.; Tuma, Roman

    2014-01-01

    Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ70 or σ54, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ54 version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ70 and σ54, the domain movements of the latter have evolved to require an activator ATPase. PMID:24553251

  10. Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

    PubMed

    Sharma, Amit; Leach, Robert N; Gell, Christopher; Zhang, Nan; Burrows, Patricia C; Shepherd, Dale A; Wigneshweraraj, Sivaramesh; Smith, David Alastair; Zhang, Xiaodong; Buck, Martin; Stockley, Peter G; Tuma, Roman

    2014-04-01

    Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase.

  11. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  12. Human DNA polymerase beta mutations allowing efficient abasic site bypass.

    PubMed

    Gieseking, Sonja; Bergen, Konrad; Di Pasquale, Francesca; Diederichs, Kay; Welte, Wolfram; Marx, Andreas

    2011-02-01

    The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.

  13. Human DNA Polymerase β Mutations Allowing Efficient Abasic Site Bypass*

    PubMed Central

    Gieseking, Sonja; Bergen, Konrad; Di Pasquale, Francesca; Diederichs, Kay; Welte, Wolfram; Marx, Andreas

    2011-01-01

    The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights. PMID:21107011

  14. A Practical Polymerase Chain Reaction Laboratory for Introductory Biology Classes.

    ERIC Educational Resources Information Center

    Bowlus, R. David; Grether, Susan C.

    1996-01-01

    Presents a polymerase chain reaction (PCR) laboratory exercise that can be performed by introductory biology students in 1 45- to 55-minute class period. Includes a general description of the polymerase chain reaction, materials needed, procedure, and details of interest to teachers. (JRH)

  15. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  16. The RNA Polymerase of Marine Cyanophage Syn5*

    PubMed Central

    Zhu, Bin; Tabor, Stanley; Raytcheva, Desislava A.; Hernandez, Alfredo; King, Jonathan A.; Richardson, Charles C.

    2013-01-01

    A single subunit DNA-dependent RNA polymerase was identified and purified to apparent homogeneity from cyanophage Syn5 that infects the marine cyanobacteria Synechococcus. Syn5 is homologous to bacteriophage T7 that infects Escherichia coli. Using the purified enzyme its promoter has been identified by examining transcription of segments of Syn5 DNA and sequencing the 5′-termini of the transcripts. Only two Syn5 RNAP promoters, having the sequence 5′-ATTGGGCACCCGTAA-3′, are found within the Syn5 genome. One promoter is located within the Syn5 RNA polymerase gene and the other is located close to the right genetic end of the genome. The purified enzyme and its promoter have enabled a determination of the requirements for transcription. Unlike the salt-sensitive bacteriophage T7 RNA polymerase, this marine RNA polymerase requires 160 mm potassium for maximal activity. The optimal temperature for Syn5 RNA polymerase is 24 °C, much lower than that for T7 RNA polymerase. Magnesium is required as a cofactor although some activity is observed with ferrous ions. Syn5 RNA polymerase is more efficient in utilizing low concentrations of ribonucleotides than T7 RNA polymerase. PMID:23258537

  17. Molecular Evolution of Multi-subunit RNA Polymerases: Sequence Analysis

    PubMed Central

    Lane, William J.; Darst, Seth A.

    2009-01-01

    Transcription in all cellular organisms is performed by multi-subunit, DNA-dependent RNA polymerases that synthesize RNA from DNA templates. Previous sequence and structural studies have elucidated the importance of shared regions common to all multi-subunit RNA polymerases. In addition RNA polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. We have created comprehensive multiple sequence alignments using all available sequence data for the multi-subunit RNA polymerase large subunits, including the bacterial β and β′ subunits and their homologues from archaebacterial RNA polymerases, the eukaryotic RNA polymerases I, II, and III, the nuclear-cytoplasmic large double-stranded DNA Virus RNA polymerases, and plant plastid RNA polymerases. In order to overcome technical difficulties inherent to the large subunit sequences, including large sequence length, small and large lineage-specific insertions, split subunits, and fused proteins, we created an automated and customizable sequence retrieval and processing system. In addition, we used our alignments to create a more expansive set of shared sequence regions and bacterial lineage-specific domain insertions. We also analyzed the intergenic gap between the bacterial β and β′ genes. PMID:19895820

  18. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  19. A general strategy for expanding polymerase function by droplet microfluidics

    PubMed Central

    Larsen, Andrew C.; Dunn, Matthew R.; Hatch, Andrew; Sau, Sujay P.; Youngbull, Cody; Chaput, John C.

    2016-01-01

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson–Crick base pairing. PMID:27044725

  20. A general strategy for expanding polymerase function by droplet microfluidics.

    PubMed

    Larsen, Andrew C; Dunn, Matthew R; Hatch, Andrew; Sau, Sujay P; Youngbull, Cody; Chaput, John C

    2016-04-05

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

  1. A third mitochondrial RNA polymerase in the moss Physcomitrella patens.

    PubMed

    Richter, Uwe; Richter, Björn; Weihe, Andreas; Börner, Thomas

    2014-02-01

    In most organisms, the mitochondrial genes are transcribed by RNA polymerases related to the single-subunit RNA polymerases of bacteriophages like T3 and T7. In flowering plants, duplication(s) of the RpoTm gene coding for the mitochondrial RNA polymerase (RPOTm) led to the evolution of additional RNA polymerases transcribing genes in plastids (RPOTp) or in both mitochondria and plastids (RPOTmp). Two putative RPOTmp enzymes were previously described to be encoded by the nuclear genes RpoTmp1 and RpoTmp2 in the moss Physcomitrella patens. Here, we report on a third Physcomitrella RpoT gene. We determined the sequence of the cDNA. Comparison of the deduced amino acid sequence with sequences of plant organellar RNA polymerases suggests that this gene encodes a functional phage-type RNA polymerase. The 78 N-terminal amino acids of the putative RNA polymerase were fused to GFP and found to target the fusion protein exclusively to mitochondria in Arabidopsis protoplasts. P. patens is the only known organism to possess three mitochondrial RNA polymerases.

  2. A general strategy for expanding polymerase function by droplet microfluidics.

    PubMed

    Larsen, Andrew C; Dunn, Matthew R; Hatch, Andrew; Sau, Sujay P; Youngbull, Cody; Chaput, John C

    2016-01-01

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem.